Transgenic organisms having tetracycline-regulated transcriptional regulatory systems

ABSTRACT

Transgenic animals carrying a transgene comprising a nucleic acid molecule encoding protein useful for regulating the expression of genes in eukaryotic cells and organisms in a highly controlled manner are disclosed. In the regulatory system of the invention, transcription of a tet operator-linked nucleotide sequence is stimulated by a transcriptional activator fusion protein composed of two polypeptides, a first polypeptide which binds to tet operator sequences in the presence of tetracycline operatively linked to a second polypeptide activates transcription in eukaryotic cells. In a preferred embodiment, the transgene encoding the transcriptional activator fusion protein is integrated at a predetermined location within the chromosome of the transgenic animal.

RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. Ser. No.08/383,754, filed Feb. 3, 1995. This application is also acontinuation-in-part of U.S. Ser. No. 08/275,876, filed Jul. 15, 1994,which is a continuation-in-part of U.S. Ser. No. 08/270,637, filed Jul.1, 1994, now abandoned. This application is also a continution-in-partof U.S. Ser. No. 08/260,452, filed Jun. 14, 1994, which is acontinuation-in-part of U.S. Ser. No. 08/076,327, filed Jun. 14, 1993,now abandoned. This application is also a continution-in-part of U.S.Ser. No. 08/076,726, filed Jun. 14, 1993. The entire contents of each ofthese applications are incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] Functional analysis of cellular proteins is greatly facilitatedthrough changes in the expression level of the corresponding gene forsubsequent analysis of the accompanying phenotype. For this approach, aninducible expression system controlled by an external stimulus isdesirable. Ideally such a system would not only mediate an “on/off”status for gene expression but would also permit limited expression of agene at a defined level.

[0003] Attempts to control gene activity have been made using variousinducible eukaryotic promoters, such as those responsive to heavy metalions (Mayo et al. (1982) Cell 29:99-1108; Brinster et al. (1982) Nature296:39-42; Searle et al. (1985) Mol. Cell. Biol. 5: 1480-1489), heatshock (Nouer et al. (1991) in Heat Shock Response, e.d. Nouer, L. , CRC,Boca Raton, Fla., pp167-220) or hormones (Lee et al. (1981) Nature294:228-232; Hynes et al. (1981) Proc. Natl. Acad. Sci. USA78:2038-2042; Klock et al. (1987) Nature 2:734-736; Israel & Kaufman(1989) Nucl. Acids Res. 17:2589-2604). However, these systems havegenerally suffered from one or both of the following problems: (1) theinducer (e.g., heavy metal ions, heat shock or steroid hormones) evokespleiotropic effects, which can complicate analyses, and (2) manypromoter systems exhibit high levels of basal activity in thenon-induced state, which prevents shut-off the regulated gene andresults in modest induction factors.

[0004] An approach to circumventing these limitations is to introduceregulatory elements from evolutionarily distant species such as E. coliinto higher eukaryotic cells with the anticipation that effectors whichmodulate such regulatory circuits will be inert to eukaryotic cellularphysiology and, consequently, will not elicit pleiotropic effects ineukaryotic cells. For example, the Lac repressor (lacR)/operator/inducersystem of E. coli functions in eukaryotic cells and has been used toregulate gene expression by three different approaches: (1) preventionof transcription initiation by properly placed lac operators at promotersites (Hu & Davidson (1987) Cell 48:555-566; Brown et al. (1987) Cell49:603-612; Figge et al. (1988) Cell 52:713-722; Fuerst et al. (1989)Proc. Natl. Acad Sci. USA 6:2549-2553: Deuschle et al. (1989) Proc.Natl. Acad. Sci. USA 86:5400-5405); (2) blockage of transcribing RNApolymerase II during elongation by a LacR/operator complex (Deuschle etal. (1990) Science 248:480-483); and (3) activation of a promoterresponsive to a fusion between LacR and the activation domain of herpessimples virus (HSV) virion protein 16 (VP16) (Labow et al. (1990) Mol.Cell. Biol. 10:3343-3356; Baim et al. (1991) Proc. Natl. Acad. Sci USA88:5072-5076).

[0005] In one version of the Lac system, expression of lacoperator-linked sequences is constitutively activated by a LacR-VP 16fusion protein and is turned off in the presence ofisopropyl-β-D-thiogalactopyranoside (IPTG) (Labow et al. (1990), citedsupra). In another version of the system, a lacR-VP16 variant is usedwhich binds to lac operators in the presence of IPTG, which can beenhanced by increasing the temperature of the cells (Baim et al.(1991),cited supra). The utility of these lac systems in eukaryotic cells islimited, in part, because IPTG acts slowly and inefficiently ineukaryotic cells and must be used at concentrations which approachcytotoxic levels. Alternatively, use of a temperature shift to inducegene expression is likely to elicit pleiotropic effects in the cells.Thus, there is a need for a more efficient inducible regulatory systemwhich exhibits rapid and high level induction of gene expression and inwhich the inducer is tolerated by eukaryotic cells without cytoxicity orpleiotropic effects.

[0006] Components of the tetracycline (Tc) resistance system of E. colihave also been found to function in eukaryotic cells and have been usedto regulate gene expression. For example, the Tet repressor (TetR),which binds to tet operator sequences in the absence of tetracycline andrepresses gene transcription, has been expressed in plant cells atsufficiently high concentrations to repress transcription from apromoter containing tet operator sequences (Gatz, C. et al. (1992) PlantJ. 2:397-404). However, very high intracellular concentrations of TetRare necessary to keep gene expression down-regulated in cells, which maynot be achievable in many situations, thus leading to “leakiness” in thesystem.

[0007] In other studies, TetR has been fused to the activation domain ofVP16 to create a tetracycline-controlled transcriptional activator (tTA)(Gossen, M. and Bujard, H. (1992) Proc. Natl. Acad. Sci. USA89:5547-5551). The tTA fusion protein is regulated by tetracycline inthe same manner as TetR, i.e., tTA binds to tet operator sequences inthe absence of tetracycline but not in the presence of tetracycline.Thus, in this system, in the continuous presence of Tc, gene expressionis kept off, and to induce transcription, Tc is removed.

SUMMARY OF THE INVENTION

[0008] This invention pertains to a regulatory system which utilizescomponents of the Tet repressor/operator/inducer system of prokaryotesto regulate gene expression in eukaryotic cells. In particular, thisinvention provides transgenic animals having a transgene comprising apolynucleotide sequence encoding a fusion protein which activatestranscription, the fusion protein comprising a first polypeptide whichbinds to a tet operator sequence in the presence of tetracycline or atetracycline analogue operatively linked to a second polypeptide whichactivates transcription in eukaryotic cells.

[0009] Preferably, the first polypeptide of the fusion protein is amutated Tet repressor, e.g., a mutated Tet repressor has at least oneamino acid substitution compared to a wild-type Tet repressor. In apreferred embodiment, the mutated Tet repressor is a mutatedTn10-derived Tet repressor having an amino acid substitution at at leastone amino acid position selected from the group consisting of position71, position 95, position 101 and position 102. Most preferably, themutated Tn10-derived Tet repressor comprises an amino acid sequenceshown in positions 1 to 207 of SEQ ID NO: 2.

[0010] The second polypeptide of the fusion protein comprises atranscriptional activation domain, such as a transcription activationdomain of herpes simplex virion protein 16.

[0011] A transgenic animal of the invention can further have a secondtransgene comprising a gene of interest operably linked to at least onetet operator sequence. Additionally, the transgenic animal can have athird transgene, e.g., comprising a polynucleotide sequence encoding afusion protein which inhibits transcription, the fusion proteincomprising a first polypeptide which binds to a tet operator sequence,operatively linked to a heterologous second polypeptide which inhibitstranscription in eukaryotic cells. The inhibitor fusion proteinpreferably binds to the tet operator sequence in the absence, but notthe presence, of tetracycline.

[0012] The transgenic animal of the invention can be, for example, amouse. In other embodiment, the animal is a cow, a goat, a sheep or apig.

[0013] The transgene(s) of the invention can be integrated randomly orat a predetermined location within the genome of the animal.

[0014] The invention further provides a method for modulatingtranscription of a tet operator-linked transgene in an animal of theinvention, involving administering tetracycline or a tetracyclineanalogue to the animal.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015]FIG. 1 is a bar graph depicting the stimulation of luciferaseactivity in HR5-C11 cells by tetracycline and different tetracyclineanalogues (1 μg/ml fc.). Cells were grown in the absence (−) or presenceof the indicated tetracyclines for 3 days before luciferase activity wasdetermined. Each solid and hatched bar represents the luciferaseactivity of a single culture dish

[0016]FIG. 2 is a graph depicting the relative luciferase activity inHR5-C11 cells when incubated with different concentrations ofdoxycycline. The results of three independent experiments are shown.

[0017]FIG. 3 is a graph depicting the kinetics of induction ofluciferase activity in HR5-C11 cells by doxycycline. HR5-C11cultureswere exposed to 1 μg/ml of doxycycline and luciferase activity wasmeasured after different time intervals; () cultures containingdoxycycline, (∘) cultures grown in the absence of antibiotic.

[0018]FIG. 4 shows the amino acid sequences of various classes of Tetrepressors, illustrating the homology between the amino acid sequencesof different classes of Tet repressors, as compared to class B Tetrepressors (e.g., Tn10-derived). Amino acid positions in other classesof Tet repressors that are identical to class B are indicated by a dash.

[0019]FIG. 5 shows the nucleotide sequences of tet operators ofdifferent classes: class A (SEQ ID NO: 11), class B (SEQ ID NO: 12),class C (SEQ ID NO: 13), class D (SEQ ID NO: 14) and class E (SEQ ID NO:15).

[0020]FIG. 6 is a schematic diagram of a bidirectional promoterconstruct for coordinate regulation of two genes of interest operativelylinked to the same tet operators for regulation by atetracycline-regulated transcriptional activator.

[0021]FIG. 7A (SEQ ID NO: 6) shows the nucleotide sequence of abidirectional promoter region for coordinate regulation of two genes ofinterest by a tetracycline-regulated transcriptional activator.

[0022]FIG. 7B (SEQ ID NO: 7) shows the nucleotide sequence of abidirectional promoter region for coordinate regulation of two genes ofinterest by a tetracycline-regulated transcriptional activator.

[0023]FIG. 8 is two graphs depicting coordinate expression of luciferaseand β-galactosidase activity by a tetracycline-regulated transcriptionalactivator.

[0024] FIGS. 9A-B are schematic diagrams of self-regulating promotersfor expression of tetracycline-regulated transcriptional activators(tTA). Panel A illustrates self-regulation of expression of a wild-typeTet repressor-containing transactivator fusion protein that binds to tetoperators in the absence of Tc. Panel B illustrates self-regulation ofexpression of a mutated Tet repressor-containing transactivator fusionprotein that binds to tet operators in the presence of Tc.

[0025]FIG. 10 is a schematic diagram of the negative and positiveregulation of a tet operator (tetOwt)-linked gene of interest by atetracycline-regulated transcriptional inhibitor protein (tSD) and atetracycline-inducible transcriptional activator fusion protein (rtTA),respectively, in the presence of increasing concentrations of thetetracycline analogue doxycycline.

[0026]FIG. 11 is a schematic diagram of the construction ofTetR-silencer domain fusion contructs by in-frame fusion of nucleic acidencoding either a Krueppel or v-erbA silencer domain to the 3′ end ofnucleic acid encoding a Tet repressor (tetr gene).

[0027]FIG. 12 is a graphic representation of the expression ofluciferase activity in mice transgenic for the luciferase reporter genealone (checked columns at right) or double transgenic animals carryingthe luciferase reporter gene and a tTAR transgene, either in the absenceof doxycycline (dark columns in middle) or in the presence ofdoxycycline (light columns at left).

[0028]FIG. 13 is a graphic representation of luciferase activity incells cotransfected with a tTA₄ transactivator construct and a tetO_(C4)luciferase reporter gene in the presence or absence of doxycycline(first pair of columns), a tTA^(R) ₆ transactivator construct and atetO_(C6) luciferase reporter gene in the presence or absence ofdoxycycline (second pair of columns), tTA₄ and tTA^(R) ₆ transactivatorconstructs and tetO_(C4) luciferase reporter gene in the presence orabsence of doxycycline (third pair of columns) or tTA₄ and tTA^(R) ₆transactivator constructs and tetO_(C6) luciferase reporter gene in thepresence or absence of doxycycline (fourth pair of columns).

[0029]FIG. 14 is a schematic diagram of model regulatory systems forcoordinate regulation of one gene in a cell using a reverse activatorand a silencer (Model A) or independent regulation of two genes in acell using an activator and a reverse activator (Model B).

DETAILED DESCRIPTION OF THE INVENTION

[0030] This invention pertains to nucleic acid molecules and proteinswhich can be used to regulate the expression of genes in eukaryoticcells or animals in a highly controlled manner. Regulation of geneexpression by the system of the invention involves at least twocomponents: A gene which is operatively linked to a regulatory sequenceand a protein which, in either the presence or absence of an inducibleagent, binds to the regulatory sequence and either activates or inhibitstranscription of the gene. The system of the invention utilizescomponents of the Tet repressor/operator/inducer system of prokaryotesto stimulate gene expression in eukaryotic cells.

[0031] Various aspects of the invention pertain to fusion proteins whichare capable of either activating or inhibiting gene transcription whenbound to tet operator (tetO) sequences, but which bind to tet operatorsequences only in the presence or, alternatively, in the absence oftetracycline, or an analogue thereof. Thus, in a host cell,transcription of a gene operatively linked to a tet operator sequence(s)is stimulated or inhibited by a fusion protein of the invention byaltering the concentration of tetracycline (or analogue) in contact withthe host cell (e.g., adding or removing tetracycline from a culturemedium, or administering or ceasing to administer tetracycline to a hostorganism, etc.).

[0032] The invention further pertains to target transcription units forregulation by the fusion protein of the invention. In addition toallowing for regulation of a single tet-operator linked gene ofinterest, the invention also provides novel transcription unitscontaining two or more genes to be transcribed that can be regulated ineither a coordinate or independent manner by a transactivator fusionprotein of the invention. Methods for stimulating or inhibitingtranscription of a gene using tetracycline (or analogues thereof), andkits which contain the components of the regulatory system describedherein, are also encompassed by the invention.

[0033] In the following subsections, the nucleic acids and proteinscomprising the components of the inducible regulatory system of theinvention, and their interrelationship, are discussed in greater detail.The subsections are as follows:

[0034] I. Tetracycline-Inducible Transcriptional Activators

[0035] A. The First Polypeptide of the Transactivator Fusion Protein

[0036] B. The Second Polypeptide of the Transactivator Fusion Protein

[0037] C. A Third Polypeptide of the Transactivator Fusion Protein

[0038] II. Expression of a Transactivator Fusion Protein

[0039] A. Expression Vectors

[0040] B. Host Cells

[0041] C. Introduction of Nucleic Acid into Host Cells

[0042] D. Transgenic Organisms

[0043] E. Homologous Recombinant Organisms

[0044] III. Target Transcription Units Regulated by aTetracycline-Inducible Transactivator

[0045] A. Regulation of Expression of tet Operator-linked NucleotideSequences

[0046] B. Coordinate Regulation of Two Nucleotide Sequences

[0047] C. Independent Regulation of Mulitple Nucleotide Sequences

[0048] D. Combined Coordinate and Independent Regulation of MultipleNucleotide Sequences

[0049] IV. Tetracycline-Regulated Transcriptional Inhibitors

[0050] A. The First Polypeptide of the Transcritional Inhibitor FusionProtein

[0051] B. The Second Polypeptide of the Transcritional Inhibitor FusionProtein

[0052] C. A Third Polypeptide of the Transcritional Inhibitor FusionProtein

[0053] D. Expression of the Transcriptional Inhibitor Fusion Protein

[0054] V. Kits of the Invention

[0055] VI. Regulation of Gene Expression by Tetracycline or AnaloguesThereof

[0056] A. Stimulation of Gene Expression by Transactivator FusionProteins

[0057] B. Inhibition of Gene Expression by Transcriptional InhibitorFusion Proteins

[0058] C. Combined Positive and Negative Regulation of Gene Expression

[0059] VII. Applications of the Invention

[0060] A. Gene Therapy

[0061] B. Production of Proteins in Vitro

[0062] C. Production of Proteins in Vivo

[0063] D. Animal Models of Human Disease

[0064] E. Production of Stable Cell Lines for Cloning

[0065] I. Tetracycline-Inducible Transcriptional Activators

[0066] In the inducible regulatory system of the invention,transcription of a gene is activated by a transcriptional activatorprotein, also referred to herein simply as a transactivator. Thetransactivator of the invention is a fusion protein. One aspect of theinvention thus pertains to fusion proteins and nucleic acids (e.g., DNA)encoding fusion proteins. The term “fusion protein” is intended todescribe at least two polypeptides, typically from different sources,which are operatively linked. With regard to the polypeptides, the term“operatively linked” is intended to mean that the two polypeptides areconnected in manner such that each polypeptide can serve its intendedfunction. Typically, the two polypeptides are covalently attachedthrough peptide bonds. The fusion protein is preferably produced bystandard recombinant DNA techniques. For example, a DNA moleculeencoding the first polypeptide is ligated to another DNA moleculeencoding the second polypeptide, and the resultant hybrid DNA moleculeis expressed in a host cell to produce the fusion protein. The DNAmolecules are ligated to each other in a 5′ to 3′ orientation such that,after ligation, the translational frame of the encoded polypeptides isnot altered (i.e., the DNA molecules are ligated to each otherin-frame).

[0067] A. The First Polypeptide of the Transactivator Fusion Protein

[0068] The transactivator fusion protein of the invention is composed,in part, of a first polypeptide which binds to a tet operator sequencein the presence of tetracycline (Tc), or an analogue thereof. The firstpolypeptide of the fusion protein is preferably a mutated Tet repressor.The term “mutated Tet repressor” is intended to include polypeptideshaving an amino acid sequence which is similar to a wild-type Tetrepressor but which has at least one amino acid difference from thewild-type Tet repressor. The term “wild-type Tet repressor” is intendedto describe a protein occurring in nature which represses transcriptionfrom tet operator sequences in prokaryotic cells in the absence of Tc.The amino acid difference(s) between a mutated Tet repressor and awild-type Tet repressor may be substitution of one or more amino acids,deletion of one or more amino acids or addition of one or more aminoacids. The mutated Tet repressor of the invention has the followingfunctional properties: 1) the polypeptide can bind to a tet operatorsequence, i.e., it retains the DNA binding specificity of a wild-typeTet repressor; and 2) it is regulated in a reverse manner bytetracycline than a wild-type Tet repressor, i.e., the mutated Tetrepressor binds to a tet operator sequence only the presence of Tc (orTc analogue) rather than in the absence of Tc.

[0069] In a preferred embodiment, a mutated Tet repressor having thefunctional properties described above is created by substitution ofamino acid residues in the sequence of a wild-type Tet repressor. Forexample, as described in Example 1, a Tn10-derived Tet repressor havingamino acid substitutions at amino acid positions 71, 95, 101 and 102 hasthe desired functional properties and thus can be used as the firstpolypeptide in the transactivator fusion protein of the invention. Theamino acid sequence of this mutated Tet repressor is shown in SEQ ID NO:2 (positions 1-207). In one embodiment of the mutated Tet repressor,position 71 is mutated from glutamic acid to lysine, position 95 ismutated from aspartic acid to asparagine, position 101 is mutated fromleucine to serine and position 102 is mutated from glycine to asparticacid, although the invention is riot limited to these particularmutations. Mutation of fewer than all four of these amino acid positionsmay be sufficient to acheive a Tet repressor with the desired functionalproperties. Accordingly, a Tet repressor is preferably mutated at atleast one of these positions. Other amino acid substitutions, deletionsor additions at these or other amino acid positions which retain thedesired functional properties of the mutated Tet repressor are withinthe scope of the invention. The crystal structure of a Tetrepressor-tetracycline complex, as described in Hinrichs, W. et al.(1994) Science 264:418-420, can be used for rational design of mutatedTet repressors. Based upon this structure, amino acid position 71 islocated outside the tetracyline binding pocket, suggesting mutation atthis site may not be necessary to achieve the desired functionalproperties of a mutated Tet repressor of the invention. In contrast,amino acid positions 95, 101 and 102 are located within the conservedtetracyline binding pocket. Thus, the tetracycline binding pocket of aTet repressor may be targeted for mutation to create a mutated Tetrepressor of the invention.

[0070] Additional mutated Tet repressors for incorporation into a fusionprotein of the invention can be created according to the teachings ofthe invention. A number of different classes of Tet repressors have beendescribed, e.g., A, B, C, D and E (of which the Tn10-encoded repressoris a class B repressor). The amino acid sequences of the differentclasses of Tet repressors share a high degree of homology (i.e., 40-60%across the length of the proteins), including in the region encompassingthe above-described mutations. The amino acid sequences of variousclasses of Tet repressors are shown and compared in FIG. 4, and are alsodescribed in Tovar, K. et al. (1988) Mol. Gen. Genet. 2:76-80.Accordingly, equivalent mutations to those described above for theTn10-derived Tet repressor can be made in other classes of Tetrepressors for inclusion in a fusion protein of the invention. Forexample, amino acid position 95, which is an aspartic acid in all fiverepressor classes, can be mutated to asparagine in any class ofrepressor. Similarly, position 102, which is glycine in all fiverepressor classes, can be mutated to aspartic acid in any class ofrepressor. Additional suitable equivalent mutations will be apparent tothose skilled in the art and can be created and tested for functionalityby procedures described herein. Nucleotide and amino acid sequences ofTet repressors of the A, C, D and E classes are disclosed in Waters, S.H. et al. (1983) Nucl. Acids Res 11:6089-6105,Unger, B. et al. (1984)Gene 31: 103-108,Unger, B. et al. (1984) Nucl Acids Res. 12:7693-7703and Tovar, K. et al. (1988) Mol. Gen. Genet. 215:76-80, respectively.These wild-type sequences can be mutated according to the teachings ofthe invention for use in the inducible regulatory system describedherein.

[0071] Alternative to the above-described mutations, additional suitablemutated Tet repressors (i.e., having the desired functional propertiesdescribed above) can be created by mutagenesis of a wild type Tetrepressor and selection as described in Example 1. The nucleotide andamino acid sequences of wild-type class B Tet repressors are disclosedin Hillen, W. and Schollmeier, K. (1983) Nucl. Acids Res. 11:525-539 andPostle, K. et al. (1984) Nucl. Acids Res. 12:4849-4863. The nucleotideand amino acid sequences of wild-type class A, C, D and E typerepressors are cited above. A mutated Tet repressor can be created andselected, for example as follows: a nucleic acid (e.g., DNA) encoding awild-type Tet repressor is subjected to random mutagenesis and theresultant mutated nucleic acids are incorporated into an expressionvector and introduced into a host cell for screening. A screening assayis used which allows for selection of a Tet repressor which binds to atet operator sequence only in the presence of tetracycline. For example,a library of mutated nucleic acids in an expression vector can beintroduced into an E. coli strain in which tet operator sequencescontrol the expression of a gene encoding a Lac repressor and the Lacrepressor controls the expression of a gene encoding an selectablemarker (e.g., drug resistance). Binding of a Tet repressor to tetoperator sequences in the bacteria will inhibit expression of the Lacrepressor, thereby inducing expression of the selectable marker gene.Cells expressing the marker gene are selected based upon the selectablephenotype (e.g., drug resistance). For wild-type Tet repressors,expression of the selectable marker gene will occur in the absence ofTc. A nucleic acid encoding a mutated Tet repressor is selected usingthis system based upon the ability of the nucleic acid to induceexpression of the selectable marker gene in the bacteria only in thepresence of Tc.

[0072] A first polypeptide of the transactivator fusion protein (e.g.,the mutated Tet repressor) has the property of binding specifically to atet operator sequence. Each class of Tet repressor has a correspondingtarget tet operator sequence. Accordingly, the term “tet operatorsequence” is intended to encompass all classes of tet operatorsequences, e.g. class A, B, C, D, and E. Nucleotide sequences of thesefive classes of tet operators are shown in FIG. 5 and SEQ ID NOs: 11-15,and are described in Waters, S. H. et al. (1983) cited supra, Hillen, W.and Schollenmeier, K. (1983) cited supra, Stüber, D. and Bujard, H.(1981) Proc. Natl. Acad. Sci. USA 78:167-171, Unger, B. et al. (1984)cited supra and Tovar, K. et al. (1988) cited supra. In a preferredembodiment, the mutated Tet repressor is a Tn10-encoded repressor (i.e.,class B) and the tet operator sequence is a class B tet operatorsequence. Alternatively, a mutated class A Tet repressor can be usedwith a class A tet operator sequence, and so on for the other classes ofTet repressor/operators.

[0073] Another approach for creating a mutated Tet repressor which bindsto a class A tet operator is to further mutate the already mutatedTn10-derived Tet repressor described herein (a class B repressor) suchthat it no longer binds efficiently to a class B type operator butinstead binds efficiently to a class A type operator. It has been foundthat nucleotide position 6 of class A or B type operators is thecritical nucleotide for recognition of the operator by its complimentaryrepressor (position 6 is a G/C pair in class B operators and an A/T pairin class A operators) (see Wissman et al. (1988) J. Mol. Biol.202:397-406). It has also been found that amino acid position 40 of aclass A or class B Tet repressor is the critical amino acid residue forrecognition of position 6 of the operator (amino acid position 40 is athreonine in class B repressors but is an alanine in class Arepressors). It still further has been found that substitution of Thr40of a class B repressor with Ala alters its binding specificity such thatthe repressor can now bind a class A operator (similarly, substitutionof Ala40 of a class A repressor with Thr alters its binding specificitysuch that the repressor can now bind a class B operator) (see Altschmiedet al. (1988) EMBO J. 7:4011-4017). Accordingly, one can alter thebinding specificity of the mutated Tn10-derived Tet repressor disclosedherein by additionally changing amino acid residue 40 from Thr to Ala bystandard molecular biology techniques (e.g., site directed mutagenesis).

[0074] A mutated Tet repressor having specific mutations (e.g., atpositions 71, 95, 101 and/or 102, as described above) can be created byintroducing nucleotide changes into a nucleic acid encoding a wild-typerepressor by standard molecular biology techniques, e.g. site directedmutagenesis or PCR-mediated mutagenesis using oligonucleotide primersincorporating the nucleotide mutations. Alternatively, when a mutatedTet repressor is identified by selection from a library, the mutatednucleic acid can be recovered from the library vector. To create atransactivator fusion protein of the invention, a nucleic acid encodinga mutated Tet repressor is then ligated in-frame to another nucleic acidencoding a transcriptional activation domain and the fusion construct isincorporated into a recombinant expression vector. The transactivatorfusion protein can be expressed by introducing the recombinantexpression vector into a host cell or animal.

[0075] B. The Second Polypeptide of the Transactivator Fusion Protein

[0076] The first polypeptide of the transactivator fusion protein isoperatively linked to a second polypeptide which directly or indirectlyactivates transcription in eukaryotic cells. To operatively link thefirst and second polypeptides, typically nucleotide sequences encodingthe first and second polypeptides are ligated to each other in-frame tocreate a chimeric gene encoding a fusion protein, although the first andsecond polypeptides can be operatively linked by other means thatpreserve the function of each polypeptide (e.g., chemicallycrosslinked). In a preferred embodiment, the second polypeptide of thetransactivator itself possesses transcriptional activation activity(i.e., the second polypeptide directly activates transcription). Inanother embodiment, the second polypeptide activates transcription by anindirect mechanims, through recruitment of a transcriptional activationprotein to interact with the fusion protein. Accordingly, the term “apolypeptide which activates transcription in eukaryotic cells” as usedherein is intended to include polypeptides which either directly orindirectly activates transcription.

[0077] Polypeptides which can function to activate transcription ineukaryotic cells are well known in the art. In particular,transcriptional activation domains of many DNA binding proteins havebeen described and have been shown to retain their activation functionwhen the domain is transferred to a heterologous protein. A preferredpolypeptide for use in the fusion protein of the invention is the herpessimplex virus virion protein 16 (referred to herein as VP16, the aminoacid sequence of which is disclosed in Triezenberg, S. J. et al. (1988)Genes Dev. 2:718-729). In one embodiment, about 127 of the C-terminalamino acids of VP16 are used. For example, a polypeptide having an aminoacid sequence shown in SEQ ID NO: 2 (positions 208-335) can be used asthe second polypeptide in the fusion protein. In another embodiment, atleast one copy of about 11 amino acids from the C-terminal region ofVP16 which retain transcriptional activation ability is used as thesecond polypeptide. Preferably, a dimer of this region (i.e., about 22amino acids) is used. Suitable C-terminal peptide portions of VP16 aredescribed in Seipel, K. et al. (EMBO J. (1992) 13:4961-4968). Forexample, a dimer of a peptide having an amino acid sequence shown in SEQID NO: 4 (encoded by a nucleotide sequence shown in SEQ ID NO: 3) can beused as the second polypeptide in the fusion protein.

[0078] Other polypeptides with transcriptional activation ability ineukaryotic cells can be used in the fusion protein of the invention.Transcriptional activation domains found within various proteins havebeen grouped into categories based upon similar structural features.Types of transcriptional activation domains include acidic transcriptionactivation domains, proline-rich transcription activation domains,serine/threonine-rich transcription activation domains andglutamine-rich transcription activation domains. Examples of acidictranscriptional activation domains include the VP16 regions alreadydescribed and amino acid residues 753-881 of GAL4. Examples ofproline-rich activation domains include amino acid residues 399-499 ofCTF/NF1 and amino acid residues 31-76 of AP2. Examples ofserine/threonine-rich transcription activation domains include aminoacid residues 1-427 of ITF1 and amino acid residues 2-451 of ITF2.Examples of glutamine-rich activation domains include amino acidresidues 175-269 of Oct1 and amino acid residues 132-243 of Sp1. Theamino acid sequences of each of the above described regions, and ofother useful transcriptional activation domains, are disclosed inSeipel, K. et al. (EMBO J. (1992) 13:4961-4968).

[0079] In addition to previously described transcriptional activationdomains, novel transcriptional activation domains, which can beidentified by standard techniques, are within the scope of theinvention. The transcriptional activation ability of a polypeptide canbe assayed by linking the polypeptide to another polypeptide having DNAbinding activity and determining the amount of transcription of a targetsequence that is stimulated by the fusion protein. For example, astandard assay used in the art utilizes a fusion protein of a putativetranscriptional activation domain and a GAL4 DNA binding domain (e.g.,amino acid residues 1-93). This fusion protein is then used to stimulateexpression of a reporter gene linked to GAL4 binding sites (see e.g.,Seipel, K. et al. (1992) EMBO J. 11:4961-4968 and references citedtherein).

[0080] In another embodiment, the second polypeptide of the fusionprotein indirectly activates transcription by recruiting atranscriptional activator to interact with the fusion protein. Forexample, a mutated tetR of the invention can be fused to a polypeptidedomain (e.g., a dimerization domain) capable of mediating aprotein-protein interaction with a transcriptional activator protein,such as an endogenous activator present in a host cell. It has beendemonstrated that functional associations between DNA binding domainsand transactivation domains need not be covalent (see e.g., Fields andSong (1989) Nature 340:245-247; Chien et al. (1991) Proc. Natl. AcadSci. USA 88:9578-9582; Gyuris et al. (1993) Cell 75:791-803; and Zervos,A. S. (1993) Cell 72:223-232). Accordingly, the second polypeptide ofthe fusion protein may not directly activate transcription but rathermay form a stable interaction with an endogenous polypeptide bearing acompatible protein-protein interaction domain and transactivationdomain. Examples of suitable interaction (or dimerization) domainsinclude leucine zippers (Landschulz et al. (1989) Science243:1681-1688), helix-loop-helix domains (Murre, C. et al. (1989) Cell58:537-544) and zinc finger domains (Frankel, A. D. et al. (1988)Science 240:70-73). Interaction of a dimerization domain present in thefusion protein with an endogeneous nuclear factor results in recruitmentof the transactivation domain of the nuclear factor to the fusionprotein, and thereby to a tet operator sequence to which the fusionprotein is bound.

[0081] C. A Third Polypeptide of the Transactivator Fusion Protein

[0082] In addition to a mutated Tet repressor and a transcriptionalactivation domain, a fusion protein of the invention can contain anoperatively linked third polypeptide which promotes transport of thefusion protein to a cell nucleus. Amino acid sequences which, whenincluded in a protein, function to promote transport of the protein tothe nucleus are known in the art and are termed nuclear localizationsignals (NLS). Nuclear localization signals typically are composed of astretch of basic amino acids. When attached to a heterologous protein(e.g., a fusion protein of the invention), the nuclear localizationsignal promotes transport of the protein to a cell nucleus. The nuclearlocalization signal is attached to a heterologous protein such that itis exposed on the protein surface and does not interfere with thefunction of the protein Preferably, the NLS is attached to one end ofthe protein, e.g. the N-terminus. The amino acid sequence of anon-limiting example of an NLS that can be included in a fusion proteinof the invention is shown in SEQ ID NO: 5. Preferably, a nucleic acidencoding the nuclear localization signal is spliced by standardrecombinant DNA techniques in-frame to the nucleic acid encoding thefusion protein (e.g., at the 5′ end).

[0083] The plasmid pUHD17-1 (described in further detail in Example 1),which comprises a transactivator of the invention having the nucleotidesequence shown in SEQ ID NO: 1, has been deposited on Jul. 8, 1994 underthe provisions of the Budapest Treaty at the Deutsche Sammlung VonMikroorganismen und ZellKulturen GmbH (DSM) in Braunschweig, Germany andassigned deposit number DSM 9279.

[0084] II. Expression of a Transactivator Fusion Protein

[0085] A. Expression Vectors

[0086] A nucleic acid of the invention encoding a transactivator fusionprotein, as described above, can be incorporated into a recombinantexpression vector in a form suitable for expression of the fusionprotein in a host cell. The term “in a form suitable for expression ofthe fusion protein in a host cell” is intended to mean that therecombinant expression vector includes one or more regulatory sequencesoperatively linked to the nucleic acid encoding the fusion protein in amanner which allows for transcription of the nucleic acid into mRNA andtranslation of the mRNA into the fusion protein. The term “regulatorysequence” is art-recognized and intended to include promoters, enhancersand other expression control elements (e.g., polyadenylation signals).Such regulatory sequences are known to those skilled in the art and aredescribed in Goeddel, Gene Expression Technology: Methods in Enzymology185, Academic Press, San Diego, Calif. (1990). It should be understoodthat the design of the expression vector may depend on such factors asthe choice of the host cell to be transfected and/or the amount offusion protein to be expressed.

[0087] When used in mammalian cells, a recombinant expression vector'scontrol functions are often provided by viral genetic material. Forexample, commonly used promoters are derived from polyoma, Adenovirus 2,cytomegalovirus and Simian Virus 40. Use of viral regulatory elements todirect expression of the fusion protein can allow for high levelconstitutive expression of the fusion protein in a variety of hostcells. In a preferred recombinant expression vector, the sequencesencoding the fusion protein are flanked upstream (i.e., 5′) by the humancytomegalovirus IE promoter and downstream (i.e., 3′) by an SV40 poly(A)signal. For example, an expression vector similar to that described inExample 1 can be used. The human cytomegalovirus IE promoter isdescribed in Boshart et al. (1985) Cell 41:521-530. Other ubiquitouslyexpressing promoters which can be used include the HSV-TK promoter(disclosed in McKnight et al. (1984) Cell 37:253-262) and β-actinpromoters (e.g., the human β-actin promoter as described by Ng et al.(1985) Mol. Cell. Biol. 5:2720-2732).

[0088] Alternatively, the regulatory sequences of the recombinantexpression vector can direct expression of the fusion proteinpreferentially in a particular cell type, i.e., tissue-specificregulatory elements can be used. Non-limiting examples oftissue-specific promoters which can be used include the albumin promoter(liver-specific; Pinkert et al. (1987) Genes Dev. 1:268-277),lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol.43:235-275), in particular promoters of T cell receptors (Winoto andBaltimore (1989) EMBO J. 8:729-733) and immunoglobulins (Banerji et al.(1983) Cell 33:729-740; Queen and Baltimore (1983) Cell 33:741-748),neuron-specific promoters (e.g., the neurofilament promoter; Byrne andRuddle (1989) Proc. Natl. Acad Sci. USA 86:5473-5477), pancreas-specificpromoters (Edlund et al. (1985) Science 230:912-916), and mammarygland-specific promoters (e.g., milk whey promoter; U.S. Pat. No.4,873,316 and European Application Publication No. 264,166).Developmentally-regulated promoters are also encompassed, for examplethe murine hox promoters (Kessel and Gruss (1990) Science 249:374-379)and the α-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev.3:537-546).

[0089] Alternatively, a self-regulating construct encoding atransactivator fusion protein can be created. To accomplish this,nucleic acid encoding the fusion protein is operatively linked to aminimal promoter sequence and at least one tet operator sequence. Forexample, the nucleic acid of SEQ ID NO: 1 can be linked to a promoterhaving a nucleotide sequence shown in SEQ ID NO: 8, 9 or 10 (the nucleicacids of SEQ ID NOs: 8 and 9 comprise a minimal CMV promoter and ten tetoperators; the nucleic acids of SEQ ID NO: 10 comprises a TK promoterand ten tet operators). A schematic diagram of such a self-regulatingconstruct is shown in FIG. 9B. When this nucleic acid is introduced intoa cell (e.g., in a recombinant expression vector), a small amount ofbasal transcription of the transactivator gene is likely to occur due to“leakiness”. In the presence of Tc (or analogue thereof) this smallamount of the transactivator fusion protein will bind to the tetoperator sequence(s) upstream of the nucleotide sequence encoding thetransactivator and stimulate additional transcription of the nucleotidesequence encoding the transactivator, thereby leading to etherproduction of the transactivator fusion protein in the cell. It will beappreciated by those skilled in the art that such a self-regulatingpromoter can also be used in conjunction with othertetracycline-regulated transactivators, such as the wild-type Tetrepressor fusion protein (tTA) described in Gossen, M. and Bujard, H.(1992) Proc. Natl. Acad. Sci. USA 89:5547-5551, which binds to tetoperators in the absence of Tc (as illustrated in FIG. 9A). When used inconjunction with this transactivator, self-regulated transcription ofthe nucleotide sequence encoding this transactivator is stimulated inthe absence of Tc. The plasmid pUHD15-3, which comprises nucleotidesequences encoding the tTA described in Gossen and Bujard (1992), citedsupra, operatively linked to a self-regulating promoter, has beendeposited on Jul. 8, 1994 under the provisions of the Budapest Treaty atthe Deutsche Sammlung Von Mikroorganismen und ZellKulturen GmbH (DSM) inBraunschweig, Germany and assigned deposit number DSM 9280.

[0090] In one embodiment, the recombinant expression vector of theinvention is a plasmid, such as that described in Example 1.Alternatively, a recombinant expression vector of the invention can be avirus, or portion thereof, which allows for expression of a nucleic acidintroduced into the viral nucleic acid. For example, replicationdefective retroviruses, adenoviruses and adeno-associated viruses can beused. Protocols for producing recombinant retroviruses and for infectingcells in vitro or in vivo with such viruses can be found in CurrentProtocols in Molecular Biology, Ausubel, F. M. et al. (eds.) GreenePublishing Associates, (1989), Sections 9.10-9.14 and other standardlaboratory manuals. Examples of suitable retroviruses include pLJ, pZIP,pWE and pEM which are well known to those skilled in the art. Examplesof suitable packaging virus lines include ψCrip, ψCre, ψ2 and ψAm. Thegenome of adenovirus can be manipulated such that it encodes andexpresses a transactivator fusion protein but is inactivated in terms ofits ability to replicate in a normal lytic viral life cycle. See forexample Berkner et al. (1988) BioTechniques 6:616; Rosenfeld et al.(1991) Science 252:431-434; and Rosenfeld et al. (1992) Cell 68:143-155.Suitable adenoviral vectors derived from the adenovirus strain Ad type 5d1324 or other strains of adenovirus (e.g., Ad2, Ad3, Ad7 etc.) are wellknown to those skilled in the art. Alternatively, an adeno-associatedvirus vector such as that described in Tratschin et al. (1985) Mol.Cell. Biol. 5:3251-3260 can be used to express a transactivator fusionprotein.

[0091] B. Host Cells

[0092] A fusion protein of the invention is expressed in a eukaryoticcell by introducing nucleic acid encoding the fusion protein into a hostcell, wherein the nucleic acid is in a form suitable for expression ofthe fusion protein in the host cell. For example, a recombinantexpression vector of the invention, encoding the fusion protein, isintroduced into a host cell. Alternatively, nucleic acid encoding thefusion protein which is operatively linked to regulatory sequences(e.g., promoter sequences) but without additional vector sequences canbe introduced into a host cell. As used herein, the term “host cell” isintended to include any eukaryotic cell or cell line so long as the cellor cell line is not incompatible with the protein to be expressed, theselection system chosen or the fermentation system employed.Non-limiting examples of mammalian cell lines which can be used includeCHO dhfr⁻ cells (Urlaub and Chasin (1980) Proc. Natl. Acad. Sci. USA77:4216-4220), 293 cells (Graham et al. (1977) J. Gen. Virol. 36: pp59)or myeloma cells like SP2 or NS0 (Galfre and Milstein (1981) MethEnzymol. 73(B):3-46).

[0093] In addition to cell lines, the invention is applicable to normalcells, such as cells to be modified for gene therapy purposes orembryonic cells modified to create a transgenic or homologousrecombinant animal. Examples of cell types of particular interest forgene therapy purposes include hematopoietic stem cells, myoblasts,hepatocytes, lymphocytes, neuronal cells and skin epithelium and airwayepithelium. Additionally, for transgenic or homologous recombinantanimals, embryonic stem cells and fertilized oocytes can be modified tocontain nucleic acid encoding a transactivator fusion protein. Moreover,plant cells can be modified to create transgenic plants.

[0094] The invention is broadly applicable and encompasses non-mammalianeukaryotic cells as well, including insect (e.g,. Sp. frugiperda), yeast(e.g., S. cerevisiae, S. pombe, P. pastoris, K. lactis, H. polymorpha;as generally reviewed by Fleer, R. (1992) Current Opinion inBiotechnology 3(5):486-496)), fungal and plant cells. Examples ofvectors for expression in yeast S. cerivisae include pYepSec1 (Baldari.et al., (1987) Embo J. 6:229-234), pMFa (Kujan and Herskowitz, (1982)Cell 30:933-943), pJRY88 (Schultz et al, (1987) Gene 54:113-123), andpYES2 (Invitrogen Corporation, San Diego, Calif.). The fusion proteincan be expressed in insect cells using baculovirus expression vectors(e.g., as described in O'Reilly et al. (1992) Baculovirus ExpressionVectors: A Laboratory Manual, Stockton Press). Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., SF9 cells) include the pAc series (Smith et al, (1983) Mol. Cell Biol.3:2156-2165) and the pVL series (Lucklow, V. A., and Summers, M. D.,(1989) Virology 170:31-39).

[0095] C. Introduction of Nucleic Acid into a Host Cell

[0096] Nucleic acid encoding the fusion protein can be introduced into ahost cell by standard techniques for transfecting eukaryotic cells. Theterm “transfecting” or “transfection” is intended to encompass allconventional techniques for introducing nucleic acid into host cells,including calcium phosphate co-precipitation, DEAE-dextran-mediatedtransfection, lipofection, electroporation and microinjection. Suitablemethods for transfecting host cells can be found in Sarnbrook et al(Molecular Cloning. A Laboratory Manual, 2nd Edition, Cold Spring HarborLaboratory press (1989)), and other laboratory textbooks.

[0097] The number of host cells transformed with a nucleic acid of theinvention will depend, at least in part, upon the type of recombinantexpression vector used and the type of transfection technique used.Nucleic acid can be introduced into a host cell transiently, or moretypically, for long term regulation of gene expression, the nucleic acidis stably integrated into the genome of the host cell or remains as astable episome in the host cell. Plasmid vectors introduced intomammalian cells are typically integrated into host cell DNA at only alow frequency. In order to identify these integrants, a gene thatcontains a selectable marker (e.g., drug resistance) is generallyintroduced into the host cells along with the nucleic acid of interest.Preferred selectable markers include those which confer resistance tocertain drugs, such as G418 and hygromycin. Selectable markers can beintroduced on a separate plasmid from the nucleic acid of interest or,are introduced on the same plasmid. Host cells transfected with anucleic acid of the invention (e.g., a recombinant expression vector)and a gene for a selectable marker can be identified by selecting forcells using the selectable marker. For example, if the selectable markerencodes a gene conferring neomycin resistance, host cells which havetaken up nucleic acid can be selected with G418. Cells that haveincorporated the selectable marker gene will survive, while the othercells die.

[0098] A host cell transfected with a nucleic acid encoding a fusionprotein of the invention can be further transfected with one or morenucleic acids which serve as the target for the fusion protein. Thetarget nucleic acid comprises a nucleotide sequence to be transcribedoperatively linked to at least one let operator sequence (described inmore detail in Section III below).

[0099] Nucleic acid encoding the fusion protein of the invention can beintroduced into eukaryotic cells growing in culture in vitro byconventional transfection techniques (e.g., calcium phosphateprecipitation, DEAE-dextran transfection, electroporation etc.). Nucleicacid can also be transferred into cells in vivo, for example byapplication of a delivery mechanism suitable for introduction of nucleicacid into cells in vivo, such as retroviral vectors (see e.g., Ferry, Net al. (1991) Proc. Natl. Acad. Sci. USA 88:8377-8381; and Kay, M. A. etal. (1992) Human Gene Therapy 3:641-647), adenoviral vectors (see e.g.,Rosenfeld, M. A. (1992) Cell 68:143-155; and Herz, J. and Gerard, R. D.(1993) Proc. Natl. Acad. Sci. USA 90: 2812-2816), receptor-mediated DNAuptake (see e.g., Wu, G. and Wu, C. H. (1988) J. Biol. Chem. 263:14621;Wilson et al. (1992) J. Biol. Chem. 267:963-967; and U.S. Pat. No.5,166,320), direct injection of DNA (see e.g., Acsadi et al. (1991)Nature 332:815-818; and Wolff et al. (1990) Science 247:1465-1468) orparticle bombardment (see e.g., Cheng, L. et al. (1993) Proc. Natl.Acad. Sci. USA 90:4455-4459; and Zelenin, A. V. et al. (1993) FEBSLetters 315:29-32). Thus, for gene therapy purposes, cells can bemodified in vitro and administered to a subject or, alternatively, cellscan be directly modified in vivo.

[0100] D. Transgenic Organisms

[0101] Nucleic acid a transactivator fusion protein can transferred intoa fertilized oocyte of a non-human animal to create a transgenic animalwhich expresses the fusion protein of the invention in one or more celltypes. A transgenic animal is an animal having cells that contain atransgene, wherein the transgene was introduced into the animal or anancestor of the animal at a prenatal, e.g., an embryonic, stage. Atransgene is a DNA which is integrated into the genome of a cell fromwhich a transgenic animal develops and which remains in the genome ofthe mature animal, thereby directing the expression of an encoded geneproduct in one or more cell types or tissues of the transgenic animal.In one embodiment, the non-human animal is a mouse, although theinvention is not limited thereto. In other embodiments, the transgenicanimal is a goat, sheep, pig, cow or other domestic farm animal. Suchtransgenic animals are useful for large scale production of proteins (socalled “gene pharming”).

[0102] A transgenic animal can be created, for example, by introducing anucleic acid encoding the fusion protein (typically linked toappropriate regulatory elements, such as a constitutive ortissue-specific enhancer) into the male pronuclei of a fertilizedoocyte, e.g., by microinjection, and allowing the oocyte to develop in apseudopregnant female foster animal. Intronic sequences andpolyadenylation signals can also be included in the transgene toincrease the efficiency of expression of the transgene. Methods forgenerating transgenic animals, particularly animals such as mice, havebecome conventional in the art and are described, for example, in U.S.Pat. Nos. 4,736,866 and 4,870,009 and Hogan, B. et al., (1986) ALaboratory Manual, Cold Spring Harbor, N.Y., Cold Spring HarborLaboratory. A transgenic founder animal can be used to breed additionalanimals carrying the transgene. Transgenic animals carrying a transgeneencoding the fusion protein of the invention can further be bred toother transgenic animals carrying other transgenes, e.g., to atransgenic animal which contains a gene operatively linked to a tetoperator sequence (discussed in more detail in Section III below).

[0103] It will be appreciated that, in addition to transgenic animals,the regulatory system described herein can be applied to othertransgeric organisms, such as transgenic plants. Transgenic plants canbe made by conventional techniques known in the art. Accordingly, theinvention encompasses non-human transgenic organisms, including animalsand plants, that contains cells which express the transactivator fusionprotein of the invention (i.e., a nucleic acid encoding thetransactivator is incorporated into one or more chromosomes in cells ofthe transgenic organism).

[0104] E. Homologous Recombinant Organisms

[0105] The invention also provides a homologous recombinant non-humanorganism expressing the fusion protein of the invention. The term“homologous recombinant organism” as used herein is intended to describean organism, e.g. animal or plant, containing a gene which has beenmodified by homologous recombination between the gene and a DNA moleculeintroduced into a cell of the animal, e.g., an embryonic cell of theanimal. In one embodiment, the non-human animal is a mouse, although theinvention is not limited thereto. An animal can be created in whichnucleic acid encoding the fusion protein has been introduced into aspecific site of the genome, i.e., the nucleic acid has homologouslyrecombined with an endogenous gene.

[0106] To create such a homologous recombinant animal, a vector isprepared which contains DNA encoding the fusion protein flanked at its5′ and 3′ ends by additional nucleic acid of a eukaryotic gene at whichhomologous recombination is to occur. The additional nucleic acidflanking that encoding the fusion protein is of sufficient length forsuccessful homologous recombination with the eukaryotic gene. Typically,several kilobases of flanking DNA (both at the 5′ and 3′ ends) areincluded in the vector (see e.g., Thomas, K. R. and Capecchi, M. R.(1987) Cell 51:503 for a description of homologous recombinationvectors). The vector is introduced into an embryonic stem cell line(e.g., by electroporation) and cells in which the introduced DNA hashomologously recombined with the endogenous DNA are selected (see e.g.,Li, E. et al. (1992) Cell 69:915). The selected cells are then injectedinto a blastocyst of an animal (e.g., a mouse) to form aggregationchimeras (see e.g., Bradley, A. in Teratocarcinomas and Embryonic StemCells: A Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987)pp. 113-152). A chimeric embryo can then be implanted into a suitablepseudopregnant female foster animal and the embryo brought to term.Progeny harbouring the homologously recombined DNA in their germ cellscan be used to breed animals in which all cells of the animal containthe homologously recombined DNA. These “germline transmission” animalscan further be mated to animals carrying a gene operatively linked to atleast one tet operator sequence (discussed in more detail in Section IIIbelow).

[0107] In addition to the homologous recombination approaches describedabove, enzyme-assisted site-specific integration systems are known inthe art and can be applied to the components of the regulatory system ofthe invention to integrate a DNA molecule at a predetermined location ina second target DNA molecule. Examples of such enzyme-assistedintegration systems include the Cre recombinase-lox target system (e.g.,as described in Baubonis, W and Sauer, B. (1993) Nucl. Acids Res.21:2025-2029; and Fukushige, S. and Sauer, B. (1992) Proc. Natl. Acad.Sci. USA 89:7905-7909) and the FLP recombinase-FRT target system (e.g.,as described in Dang, D. T. and Perrimon, N. (1992) Dev. Genet.13:367-375; and Fiering, S. et al. (1993) Proc. Natl. Acad. Sci. USA90:8469-8473).

[0108] III. Target Transcription Units Regulated by aTetracycline-Inducible Transactivator

[0109] A fusion protein of the invention is used to regulate thetranscription of a target nucleotide sequence. This target nucleotidesequence is operatively linked to a regulatory sequence to which thefusion protein binds. More specifically, the fusion protein regulatesexpression of a nucleotide sequence operatively linked to at least onetet operator sequence. Accordingly, another aspect of the inventionrelates to target nucleic acids (e.g., DNA molecules) comprising anucleotide sequence to be transcribed operatively linked to at least onetet operator sequence. Such nucleic acids are also referred to herein astet-regulated transcription units (or simply transcription units).

[0110] Within a transcription unit, the “nucleotide sequence to betranscribed” typically includes a minimal promoter sequence which is notitself transcribed but which serves (at least in part) to position thetranscriptional machinery for transcription. The minimal promotersequence is linked to the transcribed sequence in a 5′ to 3′ directionby phosphodiester bonds (i.e., the promoter is located upstream of thetranscribed sequence) to form a contiguous nucleotide sequence.Accordingly, as used herein, the terms “nucleotide sequence to betranscribed” or “target nucleotide sequence” are intended to includeboth the nucleotide sequence which is transcribed into mRNA and anoperatively linked upstream minimal promoter sequence. The term “minimalpromoter” is intended to describe a partial promoter sequence whichdefines the start site of transcription for the linked sequence to betranscribed but which by itself is not capable of initiatingtranscription efficiently, if at all. Thus, the activity of such aminimal promoter is dependent upon the binding of a transcriptionalactivator (such as the tetracycline-inducible fusion protein of theinvention) to an operatively linked regulatory sequence (such as one ormore tet operator sequences). In one embodiment, the minimal promoter isfrom the human cytomegalovirus (as described in Boshart et al. (1985)Cell 41:521-530). Preferably, nucleotide positions between about +75 to−53 and +75 to −31 are used. Other suitable minimal promoters are knownin the art or can be identified by standard techniques. For example, afunctional promoter which activates transcription of a contiguouslylinked reporter gene (e.g., chloramphenicol acetyl transferase,β-galactosidase or luciferase) can be progressively deleted until it nolonger activates expression of the reporter gene alone but ratherrequires the presence of an additional regulatory sequence(s).

[0111] Within a transcription unit, the target nucleotide sequence(including the transcribed nucleotide sequence and its upstream minimalpromoter sequence) is operatively linked to at least one tet operatorsequence. In a typical configuration, the tet operator sequence(s) isoperatively linked upstream (i.e., 5′) of the minimal promoter sequencethrough a phosphodiester bond at a suitable distance to allow fortranscription of the target nucleotide sequence upon binding of aregulatory protein (e.g., the transactivator fusion protein) to the tetoperator sequence. That is, the transcription unit is comprised of, in a5′ to 3′ direction: tet operator sequence(s)—a minimal promoter—atranscribed nucleotide sequence. It will be appreciated by those skilledin the art that there is some flexibility in the permissable distancebetween the tet operator sequence(s) and the minimal promoter, althoughtypically the tet operator sequences will be located within about200-400 base pairs upstream of the minimal promoter.

[0112] The nucleotide sequences of examples of tet-regulated promoters,containing tet operator sequences linked to a minimal promoter, that canbe used in the invention are shown in SEQ ID NO: 8-10. The nucleotidesequences of SEQ ID NOs: 8 and 9 comprise a cytomegalovirus minimalpromoter linked to ten tet operator sequences; the two nucleotidesequences differ in the distance between the operators and the firsttranscribed nucleotide. The nucleotide sequence of SEQ ID NO: 10comprises a herpes simplex virus minimal tk promoter linked to ten tetoperator sequences. The promoter of SEQ ID NO: 8 corresponds toP_(hCMV)*−1, described in Gossen, M. and Bujard, H. (1992) Proc. Natl.Acad. Sci. USA 89:5547-5551. The promoter of SEQ ID NO: 9 corresponds toP_(hCMV)*−2, also described in Gossen, M. and Bujard, H, cited supra.

[0113] Alternatively, since regulatory elements have been observed inthe art to function downstream of sequences to be transcribed, it islikely that the tet operator sequence(s) can be operatively linkeddownstream (i.e., 3′) of the transcribed nucleotide sequence. Thus, inthis configuration, the transcription unit is comprised of, in a 5′ to3′ direction: a minimal promoter—a transcribed nucleotide sequence—tetoperator sequence(s). Again, it will be appreciated that there is likelyto be some flexibility in the permissable distance downstream at whichthe tet operator sequence(s) can be linked.

[0114] The term “tet operator sequence” is intended to encompass allclasses of tet operators (e.g., A, B, C, D and E). A nucleotide sequenceto be transcribed can be operatively linked to a single tet operatorsequence, or for an enhanced range of regulation, it can be operativelylinked to multiple tet operator sequences (e.g., two, three, four, five,six, seven, eight, nine, ten or more operator sequences). In a preferredembodiment, the sequence to be transcribed is operatively linked toseven tet operator sequences.

[0115] A tet-regulated transcription unit can further be incorporatedinto a recombinant vector (e.g., a plasmid or viral vector) by standardrecombinant DNA techniques. The transcription unit, or recombinantvector in which it is contained, can be introduced into a host cell bystandard transfection techniques, such as those described above. Itshould be appreciated that, after introduction of the transcription unitinto a population of host cells, it may be necessary to select a hostcell clone which exhibit low basal expression of the tet operator-inkednucleotide sequence (i.e., selection for a host cell in which thetranscription unit has integrated at a site that results in low basalexpression of the tet operator-linked nucleotide sequence). Furthermore,a tet-regulated transcription unit can be introduced, by proceduresdescribed above, into the genome of a non-human animal at an embryonicstage or into plant cells to create a transgenic or homologousrecombinant organims carrying the transcription unit in some or all ofits cells. Again, it should be appreciated that it may be necessary toselect a transgenic or homologous organism in which there is low basalexpression of the the tet operator-linked nucleotide sequence in cellsof interest.

[0116] In one embodiment, the target nucleotide sequence of thetet-regulated transcription unit encodes a protein of interest. Thus,upon induction of transcription of the nucleotide sequence by thetransactivator of the invention and translation of the resultant mRNA,the protein of interest is produced in a host cell or animal.Alternatively, the nucleotide sequence to be transcribed can encode foran active RNA molecule, e.g., an antisense RNA molecule or ribozyme.Expression of active RNA molecules in a host cell or animal can be usedto regulate functions within the host (e.g., prevent the production of aprotein of interest by inhibiting translation of the mRNA encoding theprotein).

[0117] A transactivator of the invention can be used to regulatetranscription of an exogenous nucleotide sequence introduced into thehost cell or animal. An “exogenous” nucleotide sequence is a nucleotidesequence which is introduced into the host cell and typically isinserted into the genome of the host. The exogenous nucleotide sequencemay not be present elsewhere in the genome of the host (e.g., a foreignnucleotide sequence) or may be an additional copy of a sequence which ispresent within the genome of the host but which is integrated at adifferent site in the genome. An exogenous nucleotide sequence to betranscribed and an operatively linked tet operator sequence(s) can becontained within a single nucleic acid molecule which is introduced intothe host cell or animal.

[0118] Alternatively, a transactivator of the invention can be used toregulate transcription of an endogenous nucleotide sequence to which atet operator sequence(s) has been linked. An “endogenous” nucleotidesequence is a nucleotide sequence which is present within the genome ofthe host. An endogenous gene can be operatively linked to a tet operatorsequence(s) by homologous recombination between a tetO-containingrecombination vector and sequences of the endogeneous gene. For example,a homologous recombination vector can be prepared which includes atleast one tet operator sequence and a miminal promoter sequence flankedat its 3′ end by sequences representing the coding region of theendogenous gene and flanked at its 5′ end by sequences from the upstreamregion of the endogenous gene by excluding the actual promoter region ofthe endogenous gene. The flanking sequences are of sufficient length forsuccessful homologous recombination of the vector DNA with theendogenous gene. Preferably, several kilobases of flanking DNA areincluded in the homologous recombination vector. Upon homologousrecombination between the vector DNA and the endogenous gene in a hostcell, a region of the endogenous promoter is replaced by the vector DNAcontaining one or more tet operator sequences operably linked to aminimal promoter. Thus, expression of the endogenous gene is no longerunder the control of its endogenous promoter but rather is placed underthe control of the tet operator sequence(s) and the minimal promoter.

[0119] In another embodiment, tet operator sequences can be insertedelsewhere within an endogenous gene, preferably within a 5′ or 3′regulatory region, via homologous recombination to create an endogenousgene whose expression can be regulated by a tetracycline-regulatedfusion protein described herein. For example, one or more tetO sequencescan be inserted into a promoter or enhancer region of an endogenous genesuch that promoter or enhancer function is maintained (i.e., the tetOsequences are introduced into a site of the promoter/enhancer regionthat is not critical for promoter/enhancer function). Regions withinpromoters or enhancers which can be altered without loss ofpromoter/enhancer function are known in the art for many genes or can bedetermined by standard techniques for analyzing critical regulatoryregions. An endogenous gene having tetO sequences inserted into anon-critical regulatory region will retain the ability to be expressedin its normal constitutive and/or tissue-specific manner but,additionally, can be downregulated by a tetracycline-controlledtranscriptional inhibitor protein in a controlled manner. For example,constitutive expression of such a modified endogenous gene can beinhibited by in the presence of tetracycline (or analogue) using aninhibitor fusion protein that binds to tetO sequences in the presence oftetracycline (or analogue) (as described in further detail in Section IVand Section VI, Part B, below).

[0120] A. Regulation of Expression of tet Operator-Linked NucleotideSequences

[0121] Expression of a tet operator-linked nucleotide sequences isregulated by a transactivator fusion protein of the invention. Thus, thefusion protein and the target nucleic acid are both present in a hostcell or organism. The presence of both the transactivator fusion proteinand the target transcription unit in the same host cell or organism canbe achieved in a number of different ways. For example, a host cell canbe transfected with one nucleic acid of the expression system (e.g.,encoding the transactivator fusion protein), stably transfected cellscan be selected and then the transfected cells can be re-transfected(also referred to as “supertransfected”) with nucleic acid correspondingto the other nucleic acid of the expression system (e.g., the targetnucleic acid to be transcribed). Two distinct selectable markers can beused for selection, e.g., uptake of the first nucleic acid can beselected with G418 and uptake of the second nucleic acid can be selectedwith hygromycin. Alternatively, a single population of cells can betransfected with nucleic acid corresponding to both components of thesystem. Accordingly, the invention provides a nucleic acid compositioncomprising:

[0122] a first nucleic acid encoding a fusion protein which activatestranscription, the fusion protein comprising a first polypeptide whichbinds to a tet operator sequence in the presence of tetracycline or atetracycline analogue operatively linked to a second polypeptide whichactivates transcription in eukaryotic cells; and

[0123] a second nucleic acid comprising a nucleotide sequence to betranscribed operatively linked to at least one tet operator sequence.

[0124] In one embodiment, the two nucleic acids are two separatemolecules (e.g., two different vectors). In this case, a host cell iscotransfected with the two nucleic acid molecules or successivelytransfected first with one nucleic acid molecule and then the othernucleic acid molecule. In, another embodiment, the two nucleic acids arelinked (i.e., colinear) in the same molecule (e.g., a single vector). Inthis case, a host cell is transfected with the single nucleic acidmolecule.

[0125] The host cell may be a cell cultured in vitro or a cell presentin vivo (e.g., a cell targeted for gene therapy). The host cell canfurther be a fertilized ooctye, embryonic stem cell or any otherembryonic cell used in the creation of non-human transgenic orhomologous recombinant animals. Transgenic or homologous recombinantanimals which comprise both nucleic acid components of the expressionsystem can be created by introducing both nucleic acids into the samecells at an embryonic stage, or more preferably, an animal which cariesone nucleic acid component of the system in its genome is mated to ananimal which carries the other nucleic acid component of the system inits genome. Offspring which have inherited both nucleic acid componentscan then be identified by standard techniques.

[0126] B. Coordinate Regulation of Expression of Two NucleotideSequences

[0127] In addition to providing a system for the regulated expression ofa single transcribed nucleotide sequence, the invention further permitscoordinated regulation of the expression of two nucleotide sequencesoperatively linked to the same tet operator sequence(s). Accordingly,another aspect of the invention pertains to a novel tet-regulatedtranscription unit for coordinate regulation of two genes. In thistranscription unit, the same tet operator sequence(s) regulates theexpression of two operatively linked nucleotide sequences that aretranscribed in opposite directions from the common tet operatorsequence(s). Accordingly, one nucleotide sequence is operatively linkedto one side of the tet operator sequence (e.g., the 5′ end on the topstrand of DNA) and the other nucleotide sequence is operatively linkedto the opposite side of the tet operator sequence (e.g., the 3′ end onthe top strand of DNA). Additionally, it should be understood that eachnucleotide sequence to be transcribed includes an operatively linkedminimal promoter sequence which is located between the nucleotidesequence to be transcribed and the tet operator sequence(s).

[0128] A representative example of such a transcription unit isdiagrammed schematically in FIG. 6. In this vectors, the two nucleotidesequences, operatively linked to the same tet operator sequence(s), aretranscribed in opposite directions relative to the tet operatorsequence(s) (i.e., the sequences are transcribed in a divergent mannerupon activation by a transactivator fusion protein of the invention). By“transcribed in opposite directions relative to the tet operatorsequence(s)”, it is meant that the first nucleotide sequence istranscribed 5′ to 3′ from one strand of the DNA (e.g., the bottomstrand) and the second nucleotide sequence is transcribed 5′ to 3′ fromthe other stand of the DNA (e.g., the top strand), resulting inbidirectional transcription away from the tet operator sequence(s).

[0129] Accordingly, the invention provides a recombinant vector forcoordinately-regulated, bidirectional transcription of two nucleotidesequence. In one embodiment, the vector comprises a nucleotide sequencelinked by phosphodiester bonds comprising, in a 5′ to 3′ direction:

[0130] a first nucleotide sequence to be transcribed, operatively linkedto

[0131] at least one tet operator sequence, operatively linked to

[0132] a second nucleotide sequence to be transcribed,

[0133] wherein transcription of the first and second nucleotidesequences proceeds in opposite directions from the at least one tetoperator sequence(s) (i.e., the first and second nucleotide sequencesare transcribed in a divergent manner).

[0134] In another embodiment, the vector does not include the first andsecond nucleotide sequence to be transcribed but instead containscloning sites which allow for the introduction into the vector ofnucleotide sequences of interest. Accordingly, in this embodiment, thevector comprises a nucleotide sequence comprising in a 5′ to 3′direction:

[0135] a first cloning site for introduction of a first nucleotidesequence to be transcribed, operatively linked to

[0136] at least one tet operator sequence, operatively linked to

[0137] a second cloning site for introduction of a second nucleotidesequence to be transcribed,

[0138] wherein transcription of a first and second nucleotide sequenceintroduced into the vector proceeds in opposite directions from the atleast one tet operator sequence(s). It will be appreciated by thoseskilled in the art that this type of “cloning vector” may be in a formwhich also includes minimal promoter sequences such that a firstnucleotide sequence introduced into the first cloning site isoperatively linked to a first minimal promoter and a second nucleotidesequence introduced into the second cloning site is operatively linkedto a second minimal promoter. Alternatively, the “cloning vector” may bein a form which does not include minimal promoter sequences and instead,nucleotide sequences including linked minimal promoter sequences areintroduced into the cloning sites of the vector.

[0139] The term “cloning site” is intended to encompass at least onerestriction endonuclease site. Typically, multiple different restrictionendonuclease sites (e.g., a polylinker) are contained within the nucleicacid.

[0140] In yet another embodiment, the vector for coordinate,bidirectional transcription of two nucleotide sequences may contain afirst nucleotide to be transcribed, such as that encoding a detectablemarker (e.g., luciferase or β-galactosidase), and a cloning site forintroduction of a second nucleotide sequence of interest.

[0141] The nucleotide sequences of two different suitable bidirectionalpromoter regions for use in a vector for coordinate regulation of twonucleotide sequences to be transcribed, as described herein, are shownin FIGS. 7A and 7B (SEQ ID NOS: 6 and 7, respectively). In the constructof FIG. 7A, both minimal promoters present in the construct are derivedfrom a CMV promoter. In the construct of FIG. 7B, one minimal promoterpresent in the construct is derived from a CMV promoter, whereas thesecond minimal promoter is derived from a TK promoter. A plasmidpUHDG1316-8, comprising a bidirectional promoter of the invention, hasbeen deposited on July 8, 1994 under the provisions of the BudapestTreaty at the Deutsche Sammlung Von Mikroorganismen und ZellKulturenGmbH (DSM) in Braunschweig, Germany and assigned deposit number DSM9281.

[0142] The transcription unit of the invention for bidirectionaltranscription of two nucleotide sequences operatively linked to the sametet operator sequence(s) is useful for coordinating the expression ofthe two nucleotide sequences of interest. Preferably, at least one ofthe nucleotide sequences to be transcribed is a eukaryotic nucleotidesequence. In one application, the vector is used to producestoichiometric amounts of two subunits of a heterodimeric molecule inthe same cell. For example, the vector can be used produce antibodyheavy and light chains in the same cell or to produce growth factorreceptor subunits in the same cells. In another application, the vectoris used to express two gene products that cooperate in establishing aparticular cellular phenotype. In yet another application, the vector isused to coexpress an indicator function and a gene of interest, whereinthe indicator is utilized to monitor expression of the gene of interest.Thus, one of the two coordinately expressed sequences can encode a geneof interest and the other can encode a detectable marker, such as asurface marker or enzyme (e.g., β-galactosidase or luciferase) which isused for selection of cells expressing the gene of interest.

[0143] Transcription of the two coordinately-regulated nucleotidesequences can be induced by tetracycline (or an analogue thereof) by useof the Tc-inducible transcriptional activator of the invention toregulate expression of the two nucleotide sequences. Thus, in thissystem, expression of both nucleotide sequences is “off” in the absenceof Tc (or analogue), whereas expression is turned “on” by the presenceof Tc (or analogue). Alternatively, the vector for coordinate regulationof two nucleotide sequences can be used in conjunction with othertetracycline-regulated transcription factors known in the art. Forexample, a transactivator fusion protein of a wild-type Tet repressorfused to a transcriptional activation domain, which activates geneexpression in the absence of Tc (or analogue), such as the tTA describedin Gossen, M. and Bujard, H. (1992) Proc. Natl. Acad. Sci USA 89:5547-5551, can also be used in conduction with this target transcriptionunit for coordinate regulation.

[0144] C. Independent Regulation of Expression of Multiple NucleotideSequences

[0145] The invention still further permits independent and oppositeregulation of two or more nucleotide sequences to be transcribed.Accordingly, another aspect of the invention pertains to a noveltet-regulated transcription unit for independent regulation of two ormore genes. To independently regulate the expression of two nucleotidesequences to be transcribed, one nucleotide sequence is operativelylinked to a tet operator sequence(s) of one class type and the othernucleotide sequence is operatively linked to a tet operator sequence(s)of another class type. Accordingly, the invention provides at least onerecombinant vector for independent regulation of transcription of twonucleotide sequences. In one embodiment, the vector(s) comprises:

[0146] a first nucleotide sequence to be a transcribed operativelylinked to at least one tet operator sequence of a first class type; and

[0147] a second nucleotide sequence to be a transcribed operativelylinked to at least one tet operator sequence of a second class type.

[0148] (It should be understood that each nucleotide sequence to betranscribed also includes an operatively linked, upstream minimalpromoter sequence.) The two independently regulated transcription unitscan be included on a single vector, or alternatively, on two seperatevectors. The recombinant vector(s) containing the nucleotide sequencesto be transcribed can be introduced into a host cell or animal asdescribed previously.

[0149] In another embodiment, the vector(s) does not include the firstand second nucleotide sequence to be transcribed but instead containscloning sites which allow for the introduction into the vector ofnucleotide sequences of interest. Accordingly, in this embodiment, thevector(s) comprises:

[0150] a first cloning site for introduction of a first nucleotidesequence to be transcribed operatively linked to at least one tetoperator sequence of a first class type; and

[0151] a second cloning site for introduction of a second nucleotidesequence to be transcribed operatively linked to at least one tetoperator sequence of a second class type.

[0152] This cloning vector(s) may be in a form that already includesfirst and second minimal promoters operatively linked, respectively, tothe first and second cloning sites. Alternatively, nucleotide sequencesto be transcribed which include an operatively linked minimal promotercan be introduced into the cloning vector.

[0153] In yet another embodiment, the vector for independent regulationof two nucleotide sequences may contain a first nucleotide to betranscribed, such as that encoding a detectable marker or a suicidegene, operatively linked to at least one tet operator sequence of afirst class type and a cloning site for introduction of a secondnucleotide sequence of interest such that it is operatively linked to atleast one tet operator sequence of a second class type.

[0154] It will be appreciated by those skilled in the art that variouscombinations of classes of tet operator sequences can be used forindependent regulation of two nucleotide sequences. For example, thefirst tet operator sequence(s) can be of the class A type and the secondcan be of the class B type, or the first tet operator sequence can be ofthe class B type and the second can be of the class C type, etc.Preferably, one to the two tet operators used is a class B typeoperator.

[0155] Independent transcription of the first and second nucleotidesequences is regulated in a host cell by further introducing into thehost cell one or more nucleic acids encoding two differenttransactivator fusion proteins which bind independently to tet operatorsequences of different class types. The first fusion protein comprises apolypeptide which binds to a tet operator sequence in the presence oftetracycline or a tetracycline analogue, operatively linked to apolypeptide which activates transcription in eukaryotic cells (e.g., atransactivator fusion protein of the invention, such as a mutatedTn10-derived Tet repressor linked to a VP16 activation region). Thesecond fusion protein comprises a polypeptide which binds to a tetoperator sequence in the absence of tetracycline or a tetracyclineanalogue, operatively linked to a polypeptide which activatestranscription in eukaryotic cells (e.g., a wild-type Tn10-derived Tetrepressor linked to a VP16 activation region, such as the tTA describedin Gossen, M. and Bujard, H. (1992) Proc. Natl. Acad. Sci. USA89:5547-5551). In one embodiment, the first fusion protein binds to thetet operator sequence of the first class type used in the transcriptionunit and the second fusion protein binds to the tet operator sequence ofthe second class type used in the transcription unit. Alternatively, inanother embodiment, the first fusion protein binds to the second classtype of tet operator and the second fusion protein binds to the firstclass type of tet operator.

[0156] For example, the first nucleotide sequence to be transcribed maybe linked to a class A tet operator and the first fusion protein maybind to class A operators, whereas the second nucleotide sequence to betranscribed may be linked to a class B tet operator and the secondfusion protein may bind to class B operators. Thus, in this embodiment,transcription of the first nucleotide sequence is activated in thepresence of Tc (or analogue thereof) while transcription of the secondnucleotide sequence is activated in the absence of Tc (or analoguethereof). Alternatively, in another embodiment, the first fusion proteinbinds to class B operators and the second fusion protein binds to classA operators. In this case, transcription of the second nucleotidesequence is activated in the presence of Tc (or analogue thereof) whiletranscription of the first nucleotide sequence is activated in theabsence of Tc (or analogue thereof). Appropriate transactivator proteinsfor use in this system can be designed as described above in Section Iand in Gossen and Bujard (1992) cited supra. In order to inhibitheterodimerization between the two different types of Tet repressorfusion proteins present in the same cell, it may be necessary to mutatethe dimerization region of one or both of the transactivator fusionproteins. Mutations can be targeted to the C-terminal region of TetRknown to be involved in dimerization. The dimerization region has beendescribed in detail based upon the crystal structure of TetR (seeHinrichs, W. et al. (1994) Science 264:418-420).

[0157] This system allows for independent and opposite regulation of theexpression of two genes by Tc and analogues thereof. Use of different Tcanalogues as inducing agents may further allow for high, low orintermediate levels of expression of the different sequences (discussedin greater detail in Section V below). The novel transcription unit ofthe invention for independently regulating the expression of two genes,described above, can be used in situations where two gene products areto be expressed in the same cell but where it is desirable to expressone gene product while expression of the other gene product is turned“off”, and vice versa. For example, this system is particularly usefulfor expressing in the same host cell either a therapeutic gene or asuicide gene (i.e., a gene which encodes a product that can be used todestroy the cell, such as ricin or herpes simplex virus thymidinekinase). In many gene therapy situations, it is desirable to be able toexpress a gene for therapeutic purposes in a host cell but also to havethe capacity to destroy the host cell once the therapy is completed.This can be accomplished using the above-described system by linking thetherapeutic gene to one class of tet operator and the suicide gene toanother class of tet operator. Thus, expression of the therapeutic genein a host cell can be stimulated by Tc (in which case expression of thesuicide gene is absent). Then, once the therapy is complete, Tc isremoved, which turns off expression of the therapeutic gene and turns onexpression of the suicide gene in the cell.

[0158] D. Combined Coordinate and Independent Regulation of MultipleNucleotide Sequences

[0159] It is further possible to regulate the expression of fournucleotide sequences by combining the system described in Section IIIBwith the system described in Section IIIC such that two pairs ofsequences are coordinately regulated while one pair is independentlyregulated from the other pair. Accordingly, two target transcriptionunits can be designed comprising:

[0160] a first nucleic acid comprising in a 5′ to 3′ direction: a firstnucleotide sequence to be transcribed, a tet operator sequence(s) of afirst class type, and a second nucleotide sequence to be transcribed

[0161] a second nucleic acid comprising in a5′ to 3′ direction: a thirdnucleotide sequence to be transcribed, a tet operator sequence(s) of asecond class type, and a fourth nucleotide sequence to be transcribed.

[0162] Transcription of the first and second nucleotide sequences in thefirst nucleic acid proceeds in a divergent manner from the first classof tet operator sequence(s). Likewise, transcription of the third andfourth nucleotide sequences in the second nucleic acide proceeds in adivergent manner from the second class of let operator sequence(s).Thus, expression of the first and second nucleotide sequences iscoordinately regulated and expression of the third and fourth nucleotidesequences is coordinately regulated. However, expression of the firstand second sequences is independently (and oppositely) regulatedcompared to the third and fourth sequences through the use of twodifferent transactivator fusion proteins, as described above, one whichactivates transcription in the presence of Tc (or analogue thereof) andthe other which activates transcription in the absence of Tc (oranalogue thereof). One transactivator is designed to bind to a tetoperators of the first class type and the other is designed to bind to atet operators of the second class type. In other embodiments, ratherthan already containing first, second, third and/or fourth nucleotidesequences to be transcribed, these transcription units can containcloning sites which allow for the introduction of first, second, thirdand/or fourth nucleotide sequences to be transcribed.

[0163] IV. Tetracycline-Regulated Transcriptional Inhibitors

[0164] Another aspect of the invention pertains to transcriptionalinhibitor fusion proteins. The inhibitor fusion proteins of theinvention are constructed similarly to the transactivator fusionproteins of the invention (see Section I above) but instead ofcontaining a polypeptide domain that stimulates transcription ineukaryotic cells, the inhibitor fusion proteins contain a polypeptidedomain that inhibits transcription in eukaryotic cells. The inhibitorfusion proteins are used to downregulate the expression of genesoperably linked to tetO sequences. For example, when a tetO-linked geneis introduced into a host cell or animal, the level of basal,constitutive expression of the gene may vary depending upon the type ofcell or tissue in which the gene is introduced and on the site ofintegration of the gene. Alternatively, constitutive expression ofendogenous genes into which tetO sequences have been introduced may varydepending upon the strength of additional endogenous regulatorysequences in the vicinity. The inhibitor fusion proteins describedherein provide compositions that can be used to inhibit the expressionof such tetO-linked genes in a controlled manner.

[0165] In one embodiment, the inhibitor fusion protein of the inventioncomprises a first polypeptide that binds to tet operator sequences inthe absence, but not the presence, of tetracycline (Tc) or an analoguethereof operatively linked to a heterologous second polypeptide thatinhibits transcription in eukaryotic cells. In another embodiment, theinhibitor fusion protein comprises a first polypeptide that binds to tetoperator sequences in the presence, but not the absence, of tetracyclineoperatively linked to a heterologous second polypeptide that inhibitstranscription in eukaryotic cells. The term “heterologous” is intendedto mean that the second polypeptide is derived from a different proteinthan the first polypeptide. Like the transactivator fusion proteins, thetranscriptional inhibitor fusion proteins can be prepared using standardrecombinant DNA techniques as described herein.

[0166] A. The First Polypeptide of the Transcriptional Inhibitor FusionProtein

[0167] The transcriptional inhibitor fusion protein of the invention iscomposed, in part, of a first polypeptide which binds to a tet operatorsequence either (i) in the absence, but not the presence of tetracycline(Tc), or an analogue thereof, or alternatively, (ii) in the presence,but not the absence of Tc or an analogue thereof.

[0168] Preferably, in the former embodiment, the first polypeptide is awild-type Tet repressor (which binds to tet operator sequences in theabsence but not the presence of Tc). A wild-type Tet repressor of anyclass (e.g., A, B, C, D or E) may be used as the first polypeptide.Preferably, the wild-type Tet repressor is a Tn10-derived Tet repressor.The nucleotide and amino acid sequences of a wild-type Tn10-derived Tetrepressor are shown in SEQ ID NO: 16 and SEQ ID NO: 17, respectively.

[0169] Alternatively, in the latter embodiment, the first polypeptide isa mutated Tet repressor as described in Section I, part A above (whichbinds to tet operator sequences in the presence but not the absence ofTc). A mutated Tet repressor of any class (e.g., A, B, C, D or E) may beused as the first polypeptide. Preferably, the mutated Tet repressor isa Tn10-derived Tet repressor having one or more amino acid substitutionsat positions 71, 95, 101 and/or 102. The nucleotide and amino acidsequences of such a mutated Tn10-derived Tet repressor are shown in SEQID NO: 18 and SEQ ID NO: 19, respectively.

[0170] B. The Second Polypeptide of the Transcriptional Inhibitor FusionProtein

[0171] The first polypeptide of the transcriptional inhibitor fusionprotein is operatively linked to a second polypeptide which directly orindirectly inhibits transcription in eukaryotic cells. As described inSection I, above, to operatively link the first and second polypeptidesof a fusion protein, typically nucleotide sequences encoding the firstand second polypeptides are ligated to each other in-frame to create achimeric gene encoding the fusion protein. However, the first and secondpolypeptides can be operatively linked by other means that preserve thefunction of each polypeptide (e.g., chemically crosslinked). Althoughthe fusion proteins are typically described herein as having the firstpolypeptide at the amino-terminal end of the fusion protein and thesecond polypeptide at the carboxy-terminal end of the fusion protein, itwill be appreciated by those skilled in the art that the oppositeorientation (i.e., the second polypeptide at the amino-terminal end andthe first polypeptide at the carboxy-terminal end) is also contemplatedby the invention.

[0172] Proteins and polypeptide domains within proteins which canfunction to inhibit transcription in eukaryotic cells have beendescribed in the art (for reviews see, e.g., Renkawitz, R. (1990) Trendsin Genetics 6:192-197; and Herschbach, B. M. and Johnson, A. D. (1993)Annu. Rev. Cell. Biol. 9:479-509). Such transcriptional inhibitordomains have been referred to in the art as “silencing domains” or“repressor domains.” Although the precise mechanism by which many ofthese polypeptide domains inhibit transcription is not known (and theinvention is not intended to be limited by mechanism), there are severalpossible means by which repressor domains may inhibit transcription,including: 1) competitive inhibition of binding of either activatorproteins or the general transcriptional machinery, 2) prevention of theactivity of a DNA bound activator and 3) negative interference with theassembly of a functional preinitiation complex of the generaltranscription machinery. Thus, a repressor domain may have a directinhibitory effect on the transcriptional machinery or may inhibittranscription indirectly by inhibiting the activity of activatorproteins. Accordingly, the term “a polypeptide that inhibitstranscription in eukaryotic cells” as used herein is intended to includepolypeptides which act either directly or indirectly to inhibittranscription. As used herein, “inhibition” of transcription is intendedto mean a diminution in the level or amount of transcription of a targetgene compared to the level or amount of transcription prior toregulation by the transcriptional inhibitor protein. Transcriptionalinhibition may be partial or complete. The terms “silencer”, “repressor”and “inhibitor” are used interchangeably herein to describe a regulatoryprotein, or domains thereof, that can inhibit transcription.

[0173] A transcriptional “repressor” or “silencer” domain as describedherein is a polypeptide domain that retains its transcriptionalrepressor function when the domain is transferred to a heterologousprotein. Proteins which have been demonstrated to have repressor domainsthat can function when transferred to a heterologous protein include thev-erbA oncogene product (Baniahmad, A. et al. (1992) EMBO J.11:1015-1023), the thyroid hormone receptor (Baniahmad, supra), theretinoic acid receptor (Baniahmad, supra), and the Drosophila Krueppel(Kr) protein (Licht, J. D. et al. (1990) Nature 346:76-79; Sauer, F. andJäckle, H. (1991) Nature 353:563-566; Licht, J. D. et al. (1994) Mol.Cell. Biol. 14:4057-4066). Non-limiting examples of other proteins whichhave transcriptional repressor activity in eukaryotic cells include theDrosophila homeodomain protein even-skipped (eve), the S. cerevisiaeSsn6/Tup1 protein complex (see Herschbach and Johnson, supra), the yeastSIR1 protein (see Chien, et al. (1993) Cell 75:531-541), NeP1 (seeKohne, et al. (1993) J. Mol. Biol. 232:747-755), the Drosophila dorsalprotein (see Kirov, et al. (1994) Mol. Cell. Biol. 14:713-722; Jiang, etal. (1993) EMBO J. 12:3201-3209), TSF3 (see Chen, et al. (1993) Mol.Cell. Biol. 13:831-840), SF1 (see Targa, et al. (1992) Biochem. Biophys.Res. Comm. 188:416-423), the Drosophila hunchback protein (see Zhang, etal. (1992) Proc. Natl. Acad. Sci. USA 89:751 1-7515), the Drosophilaknirps protein (see Gerwin, et al. (1994) Mol. Cell. Biol.14:7899-7908), the WT1 protein (Wilm's tumor gene product) (see Anant,et al. (1994) Oncogene 9:3113-3126; Madden et al., (1993) Oncogene8:1713-1720), Oct-2.1 (see Lillycrop, et al. (1994) Mol. Cell. Biol.14:7633-7642), the Drosophila engrailed protein (see Badiani, et al.(1994) Genes Dev. 8:770-782; Han and Manley, (1993) EMBO J.12:2723-2733), E4BP4 (see Cowell and Hurst, (1994) Nucleic Acids Res.22:59-65) and ZF5 (see Numoto, et al. (1993) Nucleic Acids Res.21:3767-3775),

[0174] In a preferred embodiment, the second polypeptide of thetranscriptional inhibitor fusion protein of the invention is atranscriptional silencer domain of the Drosophila Krueppel protein. AC-terminal region having repressor activity can be used, such as aminoacids 403-466 of the native protein (see Sauer, F. and Jäckle, H.,supra). This region is referred to as C64KR. The nucleotide and aminoacid sequences of C64KR are shown in SEQ ID NO: 20 and SEQ ID NO: 21,respectively. Construction of an expression vector encoding a TetR-C64KRfusion protein is described in Example 4. Alternatively, an alanine-richamino terminal region of Kr that also has repressor activity can be usedas the second polypeptide of the fusion protein. For example, aminoacids 26-110 of Kr (see Licht, J. D. et al., (1990) supra) can be usedas the second polypeptide. Alternatively, shorter or longer polypeptidefragments encompassing either of the Kr silencer domains that stillretain full or partial inhibitor activity are also contemplated (e.g.,amino acids 62 to 92 of the N-terminal silencer domain; see Licht, etal. (1994) supra).

[0175] In another preferred embodiment, the second polypeptide of thetranscriptional inhibitor fusion protein of the invention is atranscriptional silencer domain of the v-erbA oncogene product. Thesilencer domain of v-erbA has been mapped to approximately amino acidresidues 362-632 of the native v-erbA oncogene product (see Baniahmad,et al. supra). Accordingly, a fragment encompassing this region is usedas the second polypeptide of the silencer domain. In one embodiment,amino acid residues 364-635 of the native v-erbA protein are used. Thenucleotide and amino acid sequences of this region of v-erbA are shownin SEQ ID NO: 22 and SEQ ID NO: 23, respectively. Construction of anexpression vector encoding a TetR-v-erbA fusion protein is described inExample 5. Alternatively, shorter or longer polypeptide fragmentsencompassing the v-erbA silencer region that still retain full orpartial inhibitor activity are also contemplated. For example, a.a.residues 346-639, 362-639, 346-632, 346-616 and 362-616 of v-erbA may beused. Additionally, polypeptide fragments encompassing these regionsthat have internal deletions yet still retain full or partial inhibitoractivity are encompassed by the invention, such as a.a. residues362-468/508-639 of v-erbA. Furthermore, two or more copies of thesilencer domain may be included in the fusion protein, such as twocopies of a.a. residues 362-616 of v-erbA. Suitable silencer polypeptidedomains of v-erbA are described further in Baniahmad, A. et al. (supra).

[0176] In other embodiments, other silencer domains are used.Non-limiting examples of polypeptide domains that can be used include:amino acid residues 120-410 of the thyroid hormone receptor alpha(THRα), amino acid residues 143-403 of the retinoic acid receptor alpha(RARα), amino acid residues 186-232 of knirps, the N-terminal region ofWT 1 (see Anant, supra), the N-terminal region of Oct-2.1 (seeLillycrop, supra), a 65 amino acid domain of E4BP4 (see Cowell andHurst, supra) and the N-terminal zinc finger domain of ZF5 (see Numoto,supra). Moreover, shorter or longer polypeptide fragments encompassingthese regions that still retain full or partial inhibitor activity arealso contemplated.

[0177] In addition to previously described transcriptional inhibitordomains, novel transcriptional inhibitor domains, which can beidentified by standard techniques, are within the scope of theinvention. The transcriptional inhibitor ability of a polypeptide can beassayed by: 1) constructing an expression vector that encodes the testsilencer polypeptide linked to another polypeptide having DNA bindingactivity (i.e., constructing a DNA binding domain-silencer domain fusionprotein), 2) cotransfecting this expression vector into host cellstogether with a reporter gene construct that is normally constitutivelyexpressed in the host cell and also contains binding sites for the DNAbinding domain and 3) determining the amount of transcription of thereporter gene construct that is inhibited by expression of the fusionprotein in the host cell. For example, a standard assay used in the artutilizes a fusion protein of a GAL4 DNA binding domain (e.g., amino acidresidues 1-147) and a test silencer domain. This fusion protein is thenused to inhibit expression of a reporter gene construct that containspositive regulatory sequences (that normally stimulate constitutivetranscription) and GAL4 binding sites (see e.g., Baniahmad, supra).

[0178] C. A Third Polypeptide of the Transcriptional Inhibitor FusionProtein

[0179] In addition to a Tet repressor and a transcriptional silencerdomain, a transcriptional inhibitor fusion protein of the invention cancontain an operatively linked third polypeptide which promotes transportof the fusion protein to a cell nucleus. As described for thetransactivator fusion proteins (see Section I, Part C, above), a nuclearlocalization signal can be incorporated into the transcriptionalinhibitor fusion protein.

[0180] D. Expression of the Transcriptional Inhibitor Fusion Protein

[0181] A nucleic acid molecule encoding a transcriptional inhibitorfusion protein of the invention can be incorporated into a recombinantexpression vector and introduced into a host cell to express the fusionprotein in the host cell as described in Section II, Parts A, B and C,above. Preferably, a host cell expressing a transcriptional inhibitorfusion protein of the invention also carries a tet operator-linked geneof interest (i.e., target nucleotide sequence to be transcribed).

[0182] Transgenic organisms expressing a transcriptional inhibitorfusion protein in cells thereof can be prepared as described in SectionII, Part D, above. Moreover, homologous recombinant organisms expressinga transcriptional inhibitor fusion protein in cells thereof are alsoencompassed by the invention and can be prepared as described in SectionII, Part E, above. The invention provides recombinant expression vectorssuitable for homologous recombination. In one embodiment, such anexpression vector comprises a nucleic acid molecule encoding atranscriptional inhibitor fusion protein of the invention which isflanked at its 5′ and 3′ ends by additional nucleic acid of a eukaryoticgene, the additional nucleic acid being of sufficient length forsuccessful homologous recombination with the eukaryotic gene. Vectorsand methods for creating homologous recombinant organisms that expressthe components of the regulatory system of the invention, and usestherefor, are described in further detail in U.S. patent applicationSer. No. 08/260,452. Preferably, a transgenic or homologous recombinantorganism of the invention expressing a transcriptional inhibitor fusionprotein in cells thereof also carries a tet operator-linked gene ofinterest (i.e., target nucleotide sequence to be transcribed) in cellsthereof.

[0183] V. Kits of the Invention

[0184] Another aspect of the invention pertains to kits which includethe components of the inducible regulatory system of the invention. Sucha kit can be used to regulate the expression of a gene of interest(i.e., a nucleotide sequence of interest to be transcribed) which can becloned into a target transcription unit. The kit may include nucleicacid encoding a transcriptional activator fusion protein or atranscriptional inhibitor fusion protein or both. Alternatively,eukaryotic cells which have nucleic acid encoding a transactivatorand/or inhibitor fusion protein stably incorporated therein, such thatthe transactivator and/or inhibitor fusion protein are expressed in theeukaryotic cell, may be provided in the kit.

[0185] In one embodiment, the kit includes a carrier means having inclose confinement therein at least two container means: a firstcontainer means which contains a first nucleic acid (e.g., DNA) encodinga transactivator fusion protein of the invention (e.g., a recombinantexpression vector encoding a first polypeptide which binds to a tetoperator sequence in the presence of tetracycline operatively linked toa second polypeptide which activates transcription in eukaryotic cells),and a second container means which contains a second target nucleic acid(e.g., DNA) for the transactivator into which a nucleotide sequence ofinterest can be cloned. The second nucleic acid typically comprises acloning site for introduction of a nucleotide sequence to be transcribed(optionally including an operatively linked minimal promoter sequence)and at least one operatively linked tet operator sequence. The term“cloning site” is intended to encompass at least one restrictionendonuclease site. Typically, multiple different restrictionendonuclease sites (e.g., a polylinker) are contained within the nucleicacid.

[0186] To regulate expression of a nucleotide sequence of interest usingthe components of the kit, the nucleotide sequence is cloned into thecloning site of the target vector of the kit by conventional recombinantDNA techniques and then the first and second nucleic acids areintroduced into a host cell or animal. The transactivator fusion proteinexpressed in the host cell or animal then regulates transcription of thenucleotide sequence of interest in the presence of the inducing agent(Tc or analogue thereof).

[0187] Alternatively, in another embodiment, the kit includes aeukaryotic cell which is stably transfected with a nucleic acid encodinga transactivator fusion protein of the invention such that thetransactivator is expressed in the cell. Thus, rather than containingnucleic acid alone, the first container means described above cancontain a eukaryotic cell line into which the first nucleic acidencoding the transactivator has been stably introduced (e.g., by stabletransfection by a conventional method such as calcium phosphateprecipitation or electroporation, etc.). In this embodiment, anucleotide sequence of interest is cloned into the cloning site of thetarget vector of the kit and then the target vector is introduced intothe eukaryotic cell expressing the transactivator fusion protein.

[0188] Alternatively or additionally, a recombinant vector of theinvention for coordinate regulation of expression of two nucleotidesequences can also be incorporated into a kit of the invention. Thevector can be included in the kit in a form that allows for introductioninto the vector of two nucleotide sequences of interest. Thus, inanother embodiment, a kit of the invention includes 1) a first nucleicacid encoding a transactivator fusion protein of the invention (or aeukaryotic cell into which the nucleic acid has been stably introduced)and 2) a second nucleic acid comprising a nucleotide sequence comprisingin a 5′ to 3′ direction: a first cloning site for introduction of afirst nucleotide sequence of interest operatively linked to at least onetet operator sequence operatively linked to a second cloning site forintroduction of a second nucleotide sequence of interest, whereintranscription of the first and second nucleotide sequences proceeds inopposite directions from the at least one tet operator sequence.Optionally, the vector can include operatively linked minimal promotersequences. In another embodiment, the vector can be in a form thatalready contains one nucleotide sequence to be transcribed (e.g.,encoding a detectable marker such as luciferase, β-galactosidase or CAT)and a cloning site for introduction of a second nucleotide sequence ofinterest to be transcribed.

[0189] The transcription units and transactivators of the invention forindependent regulation of expression of two nucleotide sequences to betranscribed can also be incorporated into a kit of the invention. Thetarget transcription units can be in a form which allows forintroduction into the transcription units of nucleotide sequences ofinterest to be transcribed. Thus, in another embodiment, a kit of theinvention includes 1) a first nucleic acid encoding a transactivatorwhich binds to a tet operator of a first class type in the presence ofTc or an analogue thereof, 2) a second nucleic acid comprising a firstcloning site for introduction of a first nucleotide sequence to betranscribed operatively linked to at least one tet operator of a firstclass type, 3) a third nucleic acid encoding a transactivator whichbinds to a tet operator of a second class type in the absence of Tc oran analogue thereof, and 4) a fourth nucleic acid comprising a secondcloning site for introduction of a second nucleotide sequence to betranscribed operatively linked to at least one tet operator of a secondclass type. (Optionally, minimal promoter sequences are included in thesecond and fourth nucleic acids). In another embodiment, one nucleotidesequence to be transcribed (e.g., encoding a suicide gene) is alreadycontained in either the second or the fourth nucleic acid. In yetanother embodiment, the nucleic acids encoding the transactivators(e.g., the first and third nucleic acids described above) can be stablyintroduced into a eukaryotic cell line which is provided in the kit.

[0190] In yet another embodiment, a kit of the invention includes afirst container means containing a first nucleic acid encoding atranscriptional inhibitor fusion protein of the invention (e.g., thefusion protein inhibits transcription in eukaryotic cells either only inthe presence of Tc or only the absence of Tc) and a second containermeans containing a second nucleic acid comprising a cloning site forintroduction of a nucleotide sequence to be transcribed operativelylinked to at least one tet operator sequence. The kit may furtherinclude a third nucleic acid encoding a transactivator fusion proteinthat binds to tetO sequences either only in the presence of Tc or onlyin the absence of Tc. Alternatively, the first and/or third nucleicacids (i.e., encoding the inhibitor or transactivator fusion proteins)may be stably incorporated into a eukaryotic host cell which is providedin the kit.

[0191] In still another embodiment, a kit of the invention may includeat least one tetracycline or tetracycline analogue. For example, the kitmay include a container means which contains tetracycline,anhydrotetracycline, doxycycline, epioxytetracycline or othertetracycline analogue described herein.

[0192] VI. Regulation of Gene Expression by Tetracycline or AnaloguesThereof

[0193] A. Stimulation of Gene Expression by Transactivator FusionProteins

[0194] In a host cell which carries nucleic acid encoding atransactivator fusion protein of the invention and a nucleotide sequenceoperatively linked to the tet operator sequence(i.e., gene of interestto be transcribed), high level transcription of the nucleotide sequenceoperatively linked to the tet operator sequence(s) does not occur in theabsence of the inducing agent, tetracycline or analogues thereof. Thelevel of basal transcription of the nucleotide sequence may varydepending upon the host cell and site of integration of the sequence,but is generally quite low or even undetectable in the absence of Tc. Inorder to induce transcription in a host cell, the host cell is contactedwith tetracycline or a tetracycline analogue. Accordingly, anotheraspect of the invention pertains to methods for stimulatingtranscription of a nucleotide sequence operatively linked to a tetoperator sequence in a host cell or animal which expresses atransactivator fusion protein of the invention. The methods involvecontacting the cell with tetracycline or a tetracycline analogue oradministering tetracycline or a tetracycline analogue to a subjectcontaining the cell.

[0195] The term “tetracycline analogue” is intended to include compoundswhich are structurally related to tetracycline and which bind to the Tetrepressor with a K_(a) of at least about 10⁶ M⁻¹. Preferably, thetetracycline analogue binds with an affinity of about 10⁹ M⁻¹ orgreater. Examples of such tetracycline analogues include, but are notlimited to, anhydrotetracycline, doxycycline, chlorotetracycline,oxytetracycline and others disclosed by Hlavka and Boothe, “TheTetracyclines,” in Handbook of Experimental Pharmacology 78, R. K.Blackwood et al. (eds.), Springer-Verlag, Berlin-N.Y., 1985; L. A.Mitscher, “The Chemistry of the Tetracycline Antibiotics”, MedicinalResearch 9, Dekker, N.Y., 1978; Noyee Development Corporation,“Tetracycline Manufacturing Processes” Chemical Process Reviews, ParkRidge, N.J., 2 volumes, 1969; R. C. Evans, “The Technology-of theTetracyclines”, Biochemical Reference Series 1, Quadrangle Press, NewYork, 1968; and H. F. Dowling, “Tetracycline”, Antibiotic Monographs,no. 3, Medical Encyclopedia, New York York, 1955. Preferred Tc analoguesfor high level stimulation of transcription are anhydrotetracycline anddoxycycline. A Tc analogue can be chosen which has reduced antibioticactivity compared to Tc. Examples of such Tc analogues areanhydrotetracycline, epioxytetracycline and cyanotetracycline.

[0196] To induce gene expression in a cell in vitro, the cell iscontacted with Tc or a Tc analogue by culturing the cell in a mediumcontaining the compound. When culturing cells in vitro in the presenceof Tc or Tc analogue, a preferred concentration range for the inducingagent is between about 10 and about 1000 ng/ml. Tc or a Tc analogue canbe directly added to media in which cells are already being cultured, ormore preferably for high levels of gene induction, cells are harvestedfrom Tc-free media and cultured in fresh media containing Tc, or ananalogue thereof.

[0197] To induce gene expression in vivo, cells within in a subject arecontacted with Tc or a Tc analogue by administering the compound to thesubject. The term “subject” is intended to include humans and othernon-human mammals including monkeys, cows, goats, sheep, dogs, cats,rabbits, rats, mice, and transgenic and homologous recombinant speciesthereof. Furthermore, the term “subject” is intended to include plants,such as transgenic plants. When the inducing agent is administered to ahuman or animal subject, the dosage is adjusted to preferably achieve aserum concentration between about 0.05 and 1.0 μg/ml. Tc or a Tcanalogue can be administered to a subject by any means effective forachieving an in vivo concentration sufficient for gene induction.Examples of suitable modes of administration include oral administration(e.g., dissolving the inducing agent in the drinking water), slowrelease pellets and implantation of a diffusion pump. To administer Tcor a Tc analogue to a transgenic plant, the inducing agent can bedissolved in water administered to the plant.

[0198] The ability to use different Tc analogues as inducing agents inthis system allows for modulate the level of expression of a tetoperator-linked nucleotide sequence. As demonstrated in Example 2,anhydrotetracycline and doxycycline have been found to be stronginducing agents. The increase in transcription of the target sequence istypically as high as 1000- to 2000-fold, and induction factors as highas 20,000 fold can be achieved. Tetracycline, chlorotetracycline andoxytetracycline have been found to be weaker inducing agents, i.e., theincrease in transcription of a target sequence is in the range of about10-fold. Thus, an appropriate tetracycline analogue is chosen as aninducing agent based upon the desired level of induction of geneexpression. It is also possible to change the level of gene expressionin a host cell or animal over time by changing the Tc analogue used asthe inducing agent. For example, there may be situations where it isdesirable to have a strong burst of gene expression initially and thenhave a sustained lower level of gene expression. Accordingly, ananalogue which stimulates a high levels of transcription can be usedinitially as the inducing agent and then the inducing agent cars beswitched to an analogue which stimulates a lower level of transcription.Moreover, when regulating the expression of multiple nucleotidesequences (e.g., when one sequence is regulated by a one of class tetoperator sequence(s) and the other is regulated by another class of tetoperator sequence(s), as described above in Section III, Part C, above),it may be possible to independently vary the level of expression of eachsequence depending upon which transactivator fusion protein is used toregulate transcription and which Tc analogue(s) is used as the inducingagent. Different transactivator fusion proteins are likely to exhibitdifferent levels of responsiveness to Tc analogues. The level ofinduction of gene expression by a particular combination oftransactivator fusion protein and inducing agent (Tc or Tc analogue) canbe determined by techniques described herein, (e.g., see Example 2).Additionally, the level of gene expression can be modulated by varyingthe concentration of the inducing agent. Thus, the expression system ofthe invention provides a mechanism not only for turning gene expressionon or off, but also for “fine tuning” the level of gene expression atintermediate levels depending upon the type and concentration ofinducing agent used.

[0199] B. Inhibition of Gene Expression by Transcriptional InhibitorFusion Proteins

[0200] The invention also provides methods for inhibiting geneexpression using the transcriptional inhibitor fusion proteins of theinvention. These methods can be used to down-regulate basal,constitutive or tissue-specific transcription of a tetO-linked gene ofinterest. For example, a gene of interest that is operatively linked totetO sequences and additional positive regulatory elements (e.g.,consitutive or tissue-specific enhancer sequences) will be transcribedin host cells at a level that is primarily determined by the strength ofthe positive regulatory elements in the host cell. Moreover, a gene ofinterest that is operatively linked to tetO sequences and only a minimalpromoter sequence may exhibit varying degrees of basal leveltranscription depending on the host cell or tissue and/or the site ofintegration of the sequence. In a host cell containing such a targetsequence and expressing an inhibitor fusion protein of the invention,transcription of the target sequence can be down regulated in acontrolled manner by altering the concentration of Tc (or analogue) incontact with the host cell. For example, when the inhibitor fusionprotein binds to tetO in the absence of Tc, the concentration of Tc incontact with the host cell is reduced to inhibit expression of thetarget gene. Preferably, a host cell is cultured in the absence of Tc tokeep target gene expression repressed. Likewise, Tc is not administeredto a host organism to keep target gene expression repressed.Alternatively, when the inhibitor fusion protein binds to tetO in thepresence of Tc, the concentration of Tc in contact with the host cell isincreased to inhibit expression of the target gene. For example, Tc isadded to the culture medium of a host cell or Tc is administered to ahost organism to repress target gene expression.

[0201] The inhibitor fusion proteins described herein can inhibit atetO-linked gene of interest in which the tetO sequences are positioned5′ of a minimal promoter sequence (e.g., tetracycline-regulatedtranscription units as described in Section III, above). Furthermore,the inhibitor fusion protein may be used to inhibit expression of a geneof interest in which tetO-linked sequences are located 3′ of thepromoter sequence but 5′ of the transcription start site. Still further,the inhibitor fusion protein may be used to inhibit expression of a geneof interest in which tetO-linked sequences are located 3′ of thetranscription start site.

[0202] Various Tc analogues as described in Section VI, part A, above,with respect to the transactivator fusion proteins can similarly be usedto regulate the activity of the inhibitor fusion proteins. Moreover, themethods of in vitro culture with Tc (or analogue) and in vivoadministration of Tc (or analogue) described in Section VI, part A, areequally applicable to the transcriptional inhibitor fusion proteins.

[0203] C. Combined Positive and Negative Regulation of Gene Expression

[0204] In addition to regulating gene expression using either atranscriptional activator or inhibitor fusion protein alone, the twotypes of fusion proteins can be used in combination to allow for bothpositive and negative regulation of expression of one or more targetgenes in a host cell. Thus, a transcriptional inhibitor protein thatbinds to tetO either (i) in the absence, but not the presence, of Tc, or(ii) in the presence, but not the absence, of Tc, can be used incombination with a transactivator protein that binds to tetO either (i)in the absence, but not the presence, of Tc, or (ii) in the presence,but not the absence, of Tc. Transactivator proteins that bind to tetO inthe absence, but not the presence, of Tc (e.g., wild-type TetR-activatorfusion proteins) are described in further detail in U.S. Ser. No.08/076,726, U.S. Ser. No. 08/076,327 and U.S. Ser. No. 08/260,452.Transactivator fusion proteins that bind to tetO in the presence, butnot the absence, of Tc (e.g., mutated TetR-activator fusion proteins)are described herein (see Section I above) and in U.S. Ser. No.08/270,637 and U.S. Ser. No. 08/275,876. Transcriptional inhibitorfusion proteins are described herein in Section IV.

[0205] As described above in Section III, Part C, when more than oneTetR fusion protein is expressed in a host cell or organism, additionalsteps may be taken to inhibit heterodimerization between the differentTetR fusion proteins. For example, a transactivator composed of a TetRof one class may be used in combination with a transcriptional inhibitorcomposed of a TetR of a second, different class that does notheterodimerize with the first class of TetR. Alternatively, amino acidresidues of the TetR involved in dimerization may be mutated to inhibitheterodimerization. However, even if some heterodimerization betweentransactivator and inhibitor fusion proteins occurs in a host cell,sufficient amounts of homodimers should be produced to allow forefficient positive and negative regulation as described herein.

[0206] It will be appreciated by those skilled in the art that variouscombinations of activator and inhibitor proteins can be used to regulatea single tetO-linked gene of interest in both a positive and negativemanner or to regulate multiple tetO-linked genes of interest in acoordinated manner or in an independent manner using the teachingsdescribed herein. The precise regulatory components utilized will dependupon the genes to be regulated and the type of regulation desired.Several non-limiting examples of how the transactivator and inhibitorfusion proteins may be used in combination are described further below.However, many other possible combinations will be evident to the skilledartisan in view of the teachings herein and are intended to beencompassed by the invention.

[0207] In a preferred embodiment, illustrated schematically in FIG. 10,expression of a tetO-linked target gene of interest in a host cell isregulated in both a negative and positive manner by the combination ofan inhibitor fusion protein that binds to tetO in the absence, but notthe presence, of tetracycline or analogue thereof (referred to as atetracycline controlled silencing domain, or tSD) and an activatorfusion protein that binds to tetO in the presence, but not the absence,of tetracycline or analogue thereof (referred to as a reversetetracycline controlled transactivator, or rtTA). In addition to tetOsequences, the target gene is linked to a promoter, and may containother positive regulatory elements (e.g., enhancer sequences) thatcontribute to basal level, constitutive transcription of the gene in thehost cell. Binding of tSD to the tetO sequences in the absence oftetracycline or analogue (e.g., doxycycline) inhibits the basalconstitutive transcription of the gene of interest, thus keeping thegene of interest in a repressed state until gene expression is desired.When gene expression is desired, the concentration of tetracycline oranalogue (e.g., doxycycline) in contact with the host cell increased.Upon addition of the drug, tSD loses the ability to bind to tetOsequences whereas the previously unbound rtTA acquires the ability tobind to tetO sequences. The resultant binding of rtTA to the tetOsequences linked to the gene of interest thus stimulates transcriptionof the gene of interest. The level of expression may be controlled bythe concentration of tetracycline or analogue, the type of Tc analogueused, the duration of induction, etc., as described previously herein.It will be appreciated that the reverse combination of fusion proteins(i.e., the inhibitor binds in the presence but not the absence of thedrug and the activator binds in the absence but not the presence of thedrug) can also be used. In this case, expression of the gene of interestis kept repressed by contacting the host cell with the drug (e.g.,culture with Tc or analogue) and gene expression is activated by removalof the drug.

[0208] In another embodiment, the activator and inhibitor fusionproteins, as described in the previous paragraph, are used incombination to coordinately regulate, in both a positive and negativemanner, two genes of interest using the bidirectional tetO-linkedtranscription unit described in Section III, Part B above. In this case,Gene 1 and Gene 2 are linked to the same tetO sequence(s), but inopposite orientations. The inhibitor fusion protein is used to repressbasal levels of transcription of both Gene 1 and Gene2 in a coordinatemanner, whereas the transactivator fusion protein is used to stimulateexpression of Gene 1 and Gene 2 in a coordinate manner.

[0209] In yet another embodiment, the activator and inhibitor fusionproteins are used to independently regulate two or more genes ofinterest using the tetO-linked transcription units as described inSection III, Part C above. For example, in one embodiment, atransactivator fusion protein that binds to one class of tetO sequences(e.g., class A) in the presence, but not the absence of Tc or analogueis used in combination with an inhibitor fusion protein that binds to asecond, different class of tetO sequences (e.g., class B) also in thepresence, but not the absence, of Tc or analogue. In a host cellcontaining Gene 1 linked to class A tetO sequences and Gene 2 linked toclass B tetO sequences, both genes will be expressed at basal levels inthe absence of the drug, whereas expression of Gene 1 will be stimulatedupon addition of the drug and expression of Gene 2 will be repressedupon addition of the drug.

[0210] Alternatively, in another embodiment, the transactivator binds toone class of tetO sequences (e.g., class A) in the presence, but not theabsence, of Tc or analogue and the inhibitor fusion protein binds to asecond, different class of tetO sequences (e.g., class B) in the absencebut not the presence of Tc or analogue. In the host cell as described inthe previous paragraph, Gene 1 will be expressed at basal levels in theabsence of the drug and will be stimulated upon addition of the drug,whereas Gene 2 will be repressed in the absence of the drug but willhave basal levels expression upon addition of the drug. Various otherpossible combinations will be apparent to the skilled artisan.Transactivator and inhibitor fusion proteins that bind to differentclasses of tetO sequences can be prepared as described in Section I,Part A. Target transcription units comprising tetO sequences ofdifferent classes can be prepared as described in Section III, Part C.

[0211] VII. Applications of the Invention

[0212] The invention is widely applicable to a variety of situationswhere it is desirable to be able to turn gene expression on and off, orregulate the level of gene expression, in a rapid, efficient andcontrolled manner without causing pleiotropic effects or cytotoxicity.Thus, the system of the invention has widespread applicability to thestudy of cellular development and differentiation in eukaryotic cells,plants and animals. For example, expression of oncogenes can beregulated in a controlled manner in cells to study their function.Additionally, the system can be used to regulate the expression ofsite-specific recombinases, such as CRE or FLP, to thereby allow forirreversible modification of the genotype of a transgenic organism undercontrolled conditions at a particular stage of development. For example,drug resistance markers inserted into the genome of transgenic plantsthat allow for selection of a particular transgenic plant could beirreversibly removed via a Tc-regulated site specific recombinase. Otherapplications of the regulatory system of the invention include:

[0213] A. Gene Therapy

[0214] The invention may be particularly useful for gene therapypurposes, in treatments for either genetic or acquired diseases. Thegeneral approach of gene therapy involves the introduction of nucleicacid into cells such that one or more gene products encoded by theintroduced genetic material are produced in the cells to restore orenhance a functional activity. For reviews on gene therapy approachessee Anderson, W. F. (1992) Science 256:808-813; Miller, A. D. (1992)Nature 357:455-460; Friedmann, T. (1989) Science 244:1275-1281; andCournoyer, D., et al. (1990) Curr. Opin. Biotech. 1:196-208. However,current gene therapy vectors typically utilize constitutive regulatoryelements which are responsive to endogenous transcriptions factors.These vector systems do not allow for the ability to modulate the levelof gene expression in a subject. In contrast, the inducible regulatorysystem of the invention provides this ability.

[0215] To use the system of the invention for gene therapy purposes, inone embodiment, cells of a subject in need of gene therapy are modifiedto contain 1) nucleic acid encoding a transactivator fusion protein ofthe invention in a form suitable for expression of the transactivator inthe host cells and 2) a gene of interest (e.g., for therapeuticpurposes) operatively linked to a let operator sequence(s). The cells ofthe subject can be modified ex vivo and then introduced into the subjector the cells can be directly modified in vivo (methods for modificationof the cells are described above in Section II). Expression of the geneof interest in the cells of the subject is then stimulated byadministering Tc or a Tc analogue to the patient. The level of geneexpression can be varied depending upon which particular Tc analogue isused as the inducing agent. The level of gene expression can also bemodulated by adjusting the dose of the tetracycline, or analoguethereof, administered to the patient to thereby adjust the concentrationachieved in the circulation and the tissues of interest.

[0216] Moreover, in another embodiment, a transcriptional inhibitorfusion protein is used to further control the level of expression of thegene of interest. For example, the cells of the subject can be modifiedto also contain a nucleic acid encoding a transcriptional inhibitorfusion protein that binds to tetO in the absence of Tc. The nucleic acidis in a form suitable for expression of the inhibitor fusion protein inthe host cells. Thus, prior to administration of Tc (or analogue) to thesubject, the basal level of transcription of the gene of interest willbe kept silent by the inhibitor fusion protein. Upon administration ofTc, binding of the inhibitor fusion protein to tetO will be inhibitedwhereas binding of the transactivator fusion will be induced, therebystimulating transcription of the gene of interest. Such combinedpositive and negative regulation of gene expression using both atransactivator fusion protein and transcriptional inhibitor fusionprotein of the invention is illustrated schematically in FIG. 10.

[0217] Conventional detection methods known in the art, such as anenzyme linked immunosorbent assay, can be used to monitor the expressionof the regulated protein of interest in the host cells and theconcentration of Tc or Tc analogue can be varied until the desired levelof expression of the protein of interest is achieved. Accordingly,expression of a protein of interest can be adjusted according to themedical needs of an individual, which may vary throughout the lifetimeof the individual. To stop expression of the gene of interest in cellsof the subject, administration of the inducing agent is stopped. Thus,the regulatory system of the invention offers the advantage overconstitutive regulatory systems of allowing for modulation of the levelof gene expression depending upon the requirements of the therapeuticsituation.

[0218] Genes of particular interest to be expressed in cells of asubject for treatment of genetic or acquired diseases include thoseencoding adenosine deaminase, Factor VIII, Factor IX, dystrophin,β-globin, LDL receptor, CFTR, insulin, erythropoietin, anti-angiogenesisfactors, growth hormone, glucocerebrosidase, β-glucouronidase,α1-antitrypsin, phenylalanine hydroxylase, tyrosine hydroxylase,ornithine transcarbamylase, arginosuccinate synthetase, UDP-glucuronysyltransferase, apoA1, TNF, soluble TNF receptor, interleukins (e.g.,IL-2), interferons (e.g., α- or γ-IFN) and other cytokines and growthfactors. Cells types which can be modified for gene therapy purposesinclude hematopoietic stem cells, myoblasts, hepatocytes, lymphocytes,skin epithelium and airway epithelium. For further descriptions of celltypes, genes and methods for gene therapy see e.g., Wilson, J. M et al.(1988) Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano, D. et al.(1990) Proc. Natl. Acad. Sci. USA 87:6141-6145; Wolff, J. A. et al.(1990) Science 247:1465-1468; Chowdhury, J. R. et al. (1991) Science254:1802-1805; Ferry, N. et al. (1991) Proc. Natl. Acad Sci. USA88:8377-8381; Wilson, J. M. et al. (1992) J. Biol. Chem. 267:963-967;Quantin, B. et al. (1992) Proc. Natl. Acad. Sci. USA 89:2581-2584; Dai,Y. et al. (1992) Proc. Natl. Acad Sci USA 89:10892-10895; van Beusechem,V. W. et al. (1992) Proc. Natl. Acad Sci. USA 89:7640-7644; Rosenfeld,M. A. et al. (1992) Cell 68:143-155; Kay, M. A. et al. (1992) Human GeneTherapy 3:641-647; Cristiano, R. J. et al. (1993) Proc. Natl. Acad. Sci.USA 90:2122-2126; Hwu, P. et al. (1993) J. Immunol. 150:4104-4115; andHerz, J. and Gerard, R. D. (1993) Proc. Natl. Acad. Sci. USA90:2812-2816.

[0219] Gene therapy applications of particular interest in cancertreatment include overexpression of a cytokine gene (e.g., TNF-α) intumor infiltrating lymphocytes or ectopic expression of cytokines intumor cells to induce an anti-tumor immune response at the tumor site),expression of an enzyme in tumor cells which can convert a non-toxicagent into a toxic agent, expression of tumor specific antigens toinduce an anti-tumor immune response, expression of tumor suppressorgenes (e.g., p53 or Rb) in tumor cells, expression of a multidrugresistance gene (e.g., MDR1 and/or MRP) in bone marrow cells to protectthem from the toxicity of chemotherapy.

[0220] Gene therapy applications of particular interest in treatment ofviral diseases include expression of trans-dominant negative viraltransactivation proteins, such as trans-dominant negative tat and revmutants for HIV or trans-dominant ICp4 mutants for HSV (see e.g.,Balboni, P. G. et al. (1993) J. Med. Virol. 41:289-295; Liem, S. E. etal. (1993) Hum. Gene Ther. 4:625-634; Malim, M. H. et al. (1992) J. Exp.Med. 176:1197-1201; Daly, T. J. et al. (1993) Biochemistry 32:8945-8954;and Smith, C. A. et al. (1992) Virology 191:581-588), expression oftrans-dominant negative envelope proteins, such as env mutants for HIV(see e.g., Steffy, K. R. et al. (1993) J. Virol 67:1854-1859),intracellular expression of antibodies, or fragments thereof, directedto viral products (“internal immunization”, see e.g., Marasco, W. A. etal. (1993) Proc. Natl. Acad. Sci. USA 90:7889-7893) and expression ofsoluble viral receptors, such as soluble CD4. Additionally, the systemof the invention can be used to conditionally express a suicide gene incells, thereby allowing for elimination of the cells after they haveserved an intended function. For example, cells used for vaccination canbe eliminated in a subject after an immune response has been generatedthe subject by inducing expression of a suicide gene in the cells byadministering Tc or a Tc analogue to the subject.

[0221] The Tc-controlled regulatory system of the invention has numerousadvantages properties that it particularly suitable for application togene therapy. For example, the system provides an “on”/“off” switch forgene expression that allows for regulated dosaging a gene product in asubject. There are several situations in which it may be desirable to beable to provide a gene product at specific levels and/or times in aregulated manner, rather than simply expressing the gene productconstitutively at a set level. For example, a gene of interest can beswitched “on” at fixed intervals (e.g., daily, alternate days, weekly,etc.) to provide the most effective level of a gene product of interestat the most effective time. The level of gene product produced in asubject can be monitored by standard methods (e.g., direct monitoringusing an immunological assay such as ELISA or RIA or indirectly bymonitoring of a laboratory parameter dependent upon the function of thegene product of interest, e.g., blood glucose levels and the like). Thisability to turn “on” expression of a gene at discrete time intervals ina subject while also allowing for the gene to be kept “off” at othertimes avoids the need for continued administration of a gene product ofinterest at intermittent intervals. This approach avoids the need forrepeated injections of a gene product, which may be painful and/or causeside effects and would likely require continuous visits to a physician.In contrast, the system of the invention avoids these drawbacks.Moreover, the ability to turn “on” expression of a gene at discrete timeintervals in a subject allows for focused treatment of diseases whichinvolve “flare ups” of activity (e.g., many autoimmune diseases) only attimes when treatment is necessary during the acute phase when pain andsymptoms are evident. At times when such diseases are in remission, theexpression system can be kept in the “off” state.

[0222] Gene therapy applications that may particularly benefit from thisability to modulate gene expression during discrete time intervalsinclude the following non-limiting examples:

[0223] Rheumatoid arthritis—genes which encode gene products thatinhibit the production of inflammatory cytokines (e.g., TNF, IL-1 andIL-12). can be expressed in subjects. Examples of such inhibitorsinclude soluble forms of a receptor for the cytokine. Additionally oralternatively, the cytokines IL-10 and/or IL4 (which stimulate aprotective Th2-type response) can be expressed. Moreover, aglucocorticomimetic receptor (GCMR) can be expressed.

[0224] Hypopituitarism—the gene for human growth hormone can beexpressed in such subjects only in early childhood, when gene expressionis necessary, until normal stature is achieved, at which time geneexpression can be downregulated.

[0225] Wound healing/Tissue regeneration—Factors (e.g., growth factors,angiogenic factors, etc.) necessary for the healing process can beexpressed only when needed and then downregulated.

[0226] Anti-Cancer Treatments—Expression of gene products useful inanti-cancer treatment can be limited to a therapeutic phase untilretardation of tumor growth is achieved, at which time expression of thegene product can be downregulated. Possible systemic anti-cancertreatments include use of tumor infiltrating lymphocytes which expressimmunostimulatory molecules (e.g., IL-2, IL-12 and the like),angiogenesis inhibitors (PF4, IL-12, etc.), Herregulin, Leukoregulin(see PCT Publication No. WO 85/04662), and growth factors for bonemarrow support therapy, such as G-CSF, GM-CSF and M-CSF. Regarding thelatter, use of the regulatory system of the invention to express factorsfor bone marrow support therapy allows for simplified therapeuticswitching at regular intervals from chemotherapy to bone marrow supporttherapy (similarly, such an approach can also be applied to AIDStreatment, e.g., simplified switching from anti-viral treatments to bonemarrow support treatment). Furthermore, controlled local targeting ofanti-cancer treatments are also possible. For example, expression of asuicide gene by a regulator of the invention, wherein the regulatoritself is controlled by, for example, a tumor-specific promoter or aradiation-induced promoter.

[0227] In another embodiment, the regulatory system of the invention isused to express angiogenesis inhibitor(s) from within a tumor via atransgene regulated by the system of the invention. Expression ofangiogenesis inhibitors in this manner may be more efficient thansystemic administration of the inhibitor and would avoid any deleteriousside effects that might accompany systemic administration. Inparticular, restricting angiogenesis inhibitor expression to withintumors could be particularly useful in treating cancer in children stillundergoing angiogenesis associated with normal cell growth.

[0228] In another embodiment, high level regulated expression ofcytokines may represent a method for focusing a patients own immuneresponse on tumor cells. Tumor cells can be transduced to expresschemoattractant and growth promoting cytokines important in increasingan individual's natural immune response. Because the highestconcentrations of cytokines will be in the proximity of the tumor, thelikelihood of eliciting an immunological response to tumor antigens isincreased. A potential problem with this type of therapy is that thosetumor cells producing the cytokines will also be targets of the immuneresponse and therefor the source of the cytokines will be eliminatedbefore eradication of all tumor cells can be certain. To combat this,expression of viral proteins known to mask infected cells from theimmune system can be placed under regulation, along with theytokinegene(s), in the same cells. One such protein is the E19 protein fromadenovirus (see e.g., Cox, Science 247:715). This protein preventstransport of class I HLA antigens to the surface of the cell and henceprevents recognition and lysis of the cell by the host's cytotoxic Tcells. Accordingly, regulated expression of E19 in tumor cells couldshield cytokine producer cells from cytotoxic T cells during the onsetof an immune response provoked by cytokine expression. After asufficient period of time has elapsed to eradicate all tumor cells butthose expressing E19, E19 expression can be turned off, causing thesecells then to fall victim to the provoked anti-tumor immune response.

[0229] Benign prostatic hypertrophy—Similar to the above, a suicide genecan be regulated by a regulator of the invention, wherein the regulatoritself is controlled by, for example, a prostate-specific promoter.

[0230] The ability to express a suicide gene (e.g., an apoptosis gene,TK gene, etc) in a controlled manner using the regulatory system of theinvention adds to the general safety and usefulness of the system. Forexample, at the end of a desired therapy, expression of a suicide genecan be triggered to eliminate cells carrying the gene therapy vector,such as cells in a bioinert implant, cells that have disseminated beyondthe intended original location, etc.

[0231] Moreover, if a transplant becomes tumorous or has side effects,the cells can be rapidly eliminated by induction of the suicide gene.The use of more than one Tc-controlled “on”/“off” switch in one cellallows for completely independent regulation of a suicide gene comparedto regulation of a gene of therapeutic interest (as described in detailherein).

[0232] The regulatory system of the invention further offers the abilityto establish a therapeutically relevant expression level for a geneproduct of interest in a subject, in contrast to unregulatedconstitutive expression which offers no flexibility in the level of geneproduct expression that can be achieved. A physiologically relevantlevel of gene product expression can be established based on theparticular medical need of the subject, e.g., based on laboratory teststhat monitor relevant gene product levels (using methods as describedabove). In addition to the clinical examples and gene products alreadydiscussed above with gene to dosaging of the gene product, othertherapeutically relevant gene products which can be expressed at adesired level at a desired time include: Factor XIII and IX inhemophiliacs (e.g., expression can be elevated during times of risk ofinjury, such as during sports); insulin or amylin in diabetics (asneeded, depending on the state of disease in the subject, diet, etc.);erythropoietin to treat erythrocytopenia (as needed, e.g., at end-stagerenal failure); low-density lipoprotein receptor (LDLr) or verylow-density lipoprotein receptor (VLDLr) for artherosclerosis or genetherapy in liver (e.g., using ex vivo implants). Applications totreatment of central nervous system disorders are also encompassed. Forexample, in Alzheimer's disease, “fine tuned” expression of cholineacetyl transferase (ChAT) to restore acetylcholine levels, neurotrophicfactors (e.g., NGF, BDNGF and the like) and/or complement inhibitors(e.g., sCR1, sMCP, sDAF, sCD59 etc.) can be accomplished. Such geneproducts can be provided, for example, by transplanted cells expressingthe gene products in a regulated manner using the system of theinvention. Moreover, Parkinson's disease can be treated by “fine tuned”expression of tyrosine hydroxylase (TH) to increase levodopa anddopamine levels.

[0233] In addition to the proteinaceous gene products discussed above,gene products that are functional RNA molecules (such as anti-sense RNAsand ribozymes) can be expressed in a controlled manner in a subject fortherapeutic purposes. For example, a ribozyme can be designed whichdiscriminates between a mutated form of a gene and a wild-type gene.Accordingly, a “correct” gene (e.g., a wild-type p53 gene) can beintroduced into a cell in parallel with introduction of a regulatedribozyme specific for the mutated form of the gene (e.g., a mutatedendogenous p53 gene) to remove the defective mRNA expressed from theendogenous gene. This approach is particularly advantageous insituations in which a gene product from the defective gene wouldinterfere with the action of the exogenous wild-type gene.

[0234] Expression of a gene product in a subject using the regulatorysystem of the invention is modulated using tetracycline or analoguesthereof. Such drugs can be administered by any route appropriate fordelivery of the drug to its desired site of action (e.g., delivery tocells containing a gene whose expression is to be regulated). Dependingon the particular cell types involved, preferred routes ofadministration may include oral administration, intravenousadministration and topical administration (e.g., using a transdermalpatch to reach cells of a localized transplant under the skin, such askeratinocytes, while avoiding any possible side effects from systemictreatment).

[0235] In certain gene therapy situations, it may be necessary ordesirable to take steps to avoid or inhibit unwanted immune reactions ina subject receiving treatment. To avoid a reaction against the cellsexpressing the therapeutic gene product, a subject's own cells aregenerally used, when possible, to express the therapeutic gene product,either by in vivo modification of the subject's cells or by obtainingcells from the subject, modifying them ex vivo and returning them to thesubject. In situations where allogeneic or xenogeneic cells are used toexpress a gene product of interest, the regulatory system of theinvention, in addition to regulating a therapeutic gene, can also beused to regulate one or more genes involved in the immune recognition ofthe cells to inhibit an immune reaction against the foreign cells. Forexample, cell-surface molecules involved in recognition of a foreigncell by T lymphocytes can be downmodulated on the surface of a foreigncell used for delivery of a therapeutic gene product, such as byregulated expression in the foreign cell of a ribozyme which cleaves themRNA encoding the cell-surface molecule. Particularly preferred cellsurface molecules which can be downmodulated in this manner to inhibitan unwanted immune response include class I and/or class II majorhistocompatibility complex (MHC) molecules, costimulatory molecules(e.g., B7-1 and/or B7-2), CD40, and various “adhesion” molecules, suchas ICAM-1 or ICAM-2. Using approaches described herein for independentbut coordinate regulation of multiple geneses the same cell, thedown-regulation of expression of a cell-surface molecule(s) in a hostcell can be coordinated with the up-regulation of expression of atherapeutic gene. Accordingly, after therapy is completed and expressionof the therapeutic gene is halted, expression of the endogenous cellsurface molecule(s) can be restored to normal.

[0236] Furthermore, as described above regarding anti-cancer treatments,a viral protein (e.g., adenovirus E19 protein) that downmodulatesexpression of MHC antigens can be regulated in host cells using thesystem of the invention as a means of avoiding unwanted immunologicalreactions.

[0237] In addition to avoiding or inhibiting an immune response againsta foreign cell delivering a therapeutic gene product, it may also benecessary, in certain situations, to avoid or inhibit an immune responseagainst certain components of the regulatory system of the invention(e.g., the regulator fusion proteins described herein) that areexpressed in a subject, since these fusion proteins containnon-mammalian polypeptides that may stimulate an unwanted immunereaction. In this regard, regulator fusion proteins can be designedand/or selected for a decreased ability to stimulate an immune responsein a host. For example, a transcriptional activator domain for use inthe regulator fusion protein can be chosen which has minimalimmunogenicity. In this regard, a wild-type transcriptional activationdomain of the herpes simplex virus protein VP16 may not be a preferredtranscriptional activation domain for use in vivo, since it maystimulate an immune response in mammals. Alternative transcriptionalactivation domains can be used, as described herein, based on theirreduced immunogenicity in a subject. For example, a transcriptionalactivation domain of a protein of the same species as the host may bepreferred (e.g., a transcriptional activation domain from a humanprotein for use of a regulatory fusion protein in humans).Alternatively, a regulatory fusion protein of the invention can bemodified to reduce its immunogenicity in subjects, e.g., by identifyingand modifying one or more dominant T cell epitopes within a polypeptideof the fusion protein (e.g., either the Tet repressor moiety or thetranscriptional modulator moiety, such as a VP16 polypeptide). Such Tcell epitopes can be identified by standard methods and altered bymutagenesis, again by standard methods. A modified form of a regulatorfusion protein can then be selected which retains its originaltranscriptional regulatory ability yet which exhibits reducedimmunogenicity in a subject as compared to an unmodified fusion protein.

[0238] In addition to the foregoing, all conventional methods forgenerally or specifically downmodulating immune responses in subjectscan be combined with the use of the regulatory system of the inventionin situations where inhibition of immune responses is desired. Generalimmunosuppressive agents, such as cyclosporin A and/or FK506, can beadministered to the subject. Alternatively, immunomodulatory agentswhich may allow for more specific immunosuppression can be used. Suchagents may include inhibitors of costimulatory molecules (e.g., aCTLA4Ig fusion protein, soluble CD4, anti-CD4 antibodies, anti-B7-1and/or anti-B7-2 antibodies or anti-gp39 antibodies).

[0239] Finally, in certain situations, a delivery vehicle for cellsexpressing a therapeutic gene can be chosen which minimizes exposure oftransplanted cells to the immune system. For example, cells can beimplanted into bioinert capsules/biocompatible membranes with poreswhich allow for diffusion of proteins (e.g., a therapeutic gene productof interest) out of the implant and diffusion of nutrients and oxygeninto the implant but which prevent entry of immune cells, therebyavoiding exposure of the transplanted cells to the immune system (as hasbeen applied to islet cell transplantation).

[0240] B. Production of Proteins in Vitro

[0241] Large scale production of a protein of interest can beaccomplished using cultured cells in vitro which have been modified tocontain 1) a nucleic acid encoding a transactivator fusion protein ofthe invention in a form suitable for expression of the transactivator inthe cells and 2) a gene encoding the protein of interest operativelylinked to a tet operator sequence(s). For example, mammalian, yeast orfungal cells can be modified to contain these nucleic acid components asdescribed herein. The modified mammalian, yeast or fungal cells can thenbe cultured by standard fermentation techniques in the presence of Tc oran analogue thereof to induce expression of the gene and produce theprotein of interest. Accordingly, the invention provides a productionprocess for isolating a protein of interest. In the process, a host cell(e.g., a yeast or fungus), into which has been introduced both a nucleicacid encoding a transactivator fusion protein of the invention and anucleic acid encoding the protein of the interest operatively linked toat least one tet operator sequence, is grown at production scale in aculture medium in the presence of tetracycline or a tetracyclineanalogue to stimulate transcription of the nucleotides sequence encodingthe protein of interest (i.e., the nucleotide sequence operativelylinked to the tet operator sequence(s)) and the protein of interest isisolated from harvested host cells or from the culture medium. Standardprotein purification techniques can be used to isolate the protein ofinterest from the medium or from the harvested cells.

[0242] C. Production of Proteins in Vivo

[0243] The invention also provides for large scale production of aprotein of interest in animals, such as in transgenic farm animals.Advances in transgenic technology have made it possible to producetransgenic livestock, such as cattle, goats, pigs and sheep (reviewed inWall, R. J. et al. (1992) J. Cell. Biochem. 49:113-120; and Clark, A. J.et al. (1987) Trends in Biotechnology 5:20-24). Accordingly, transgeniclivestock carrying in their genome the components of the inducibleregulatory system of the invention can be constructed, wherein a geneencoding a protein of interest is operatively linked to at least one tetoperator sequence. Gene expression, and thus protein production, isinduced by administering Tc (or analogue thereof) to the transgenicanimal. Protein production can be targeted to aparticular tissue bylinking the nucleic acid encoding the transactivator fusion protein toan appropriate tissue-specific regulatory element(s) which limitsexpression of the transactivator to certain cells. For example, amammary gland-specific regulatory element, such as the milk wheypromoter (U.S. Pat. No. 4,873,316 and European Application PublicationNo. 264,166), can be linked to the transactivator transgene to limitexpression of the transactivator to mammary tissue. Thus, in thepresence of Tc (or analogue), the protein of interest will be producedin the mammary tissue of the transgenic animal. The protein can bedesigned to be secreted into the milk of the transgenic animal, and ifdesired, the protein can then be isolated from the milk.

[0244] D. Animal Models of Human Disease

[0245] The transcriptional activator and inhibitor proteins of theinvention can be used alone or in combination to stimulate or inhibitexpression of specific genes in animals to mimic the pathophysiology ofhuman disease to thereby create animal models of human disease. Forexample, in a host animal, a gene of interest thought to be involved ina disease can be placed under the transcriptional control of one or moretet operator sequences (e.g., by homologous recombination, as describedherein). Such an animal can be mated to a second animal carrying one ormore transgenes for a transactivator fusion protein and/or an inhibitorfusion protein to create progeny that carry both atetracycline-regulated fusion protein(s) gene and a tet-regulated targetsequence. Expression of the gene of interest in these progeny can bemodulated using tetracycline (or analogue). For example, expression ofthe gene of interest can be downmodulated using a transcriptionalinhibitor fusion protein to examine the relationship between geneexpression and the disease. Such an approach may be advantageous overgene “knock out” by homologous recombination to create animal models ofdisease, since the tet-regulated system described herein allows forcontrol over both the levels of expression of the gene of interest andthe timing of when gene expression is down-or up-regulated.

[0246] E. Production of Stable Cell Lines for Gene Cloning and OtherUses

[0247] The transcriptional inhibitor system described herein can be usedkeep gene expression “off” (i.e., expressed) to thereby allow productionof stable cell lines that otherwise may not be produced. For example,stable cell lines carrying genes that are cytotoxic to the cells can bedifficult or impossible to create due to “leakiness” in the expressionof the toxic genes. By repressing gene expression of such toxic genesusing the transcriptional inhibitor fusion proteins of the invention,stable cell lines carrying toxic genes may be created. Such stable celllines can then be used to clone such toxic genes (e.g., inducing theexpression of the toxic genes under controlled conditions using Tc oranalog). General methods for expression cloning of genes, to which thetranscriptional inhibitor system of the invention can be applied, areknown in the art (see e.g., Edwards, C. P. and Aruffo, A. (1993) Curr.Opin. Biotech. 4:558-563) Moreover, the transcriptional inhibitor systemcan be applied to inhibit basal expression of genes in other cells tocreate stable cell lines, such as in embryonic stem (ES) cells. Residualexpression of certain genes introduced into ES stems may result in aninability to isolate stably transfected clones. Inhibition oftranscription of such genes using the transcriptional inhibitor systemdescribed herein may be useful in overcoming this problem.

[0248] Advantages

[0249] The inducible regulatory system of the invention utilizing atransactivator fusion protein addresses and overcomes many of thelimitations of other inducible regulatory systems in the art. Forexample, very high intracellular concentrations of the transcriptionalactivator fusion protein of the invention are not required for efficientregulation of gene expression. Additionally, since gene expression isinduced by adding rather than removing the inducing agent, the inductionkinetics in the system of the invention are not limited by the rate ofremoval of the inducing agent and thus are typically faster. Moreover,the inducing agent is only present when gene transcription is induced,thereby avoiding the need for the continuous presence of an agent tokeep gene expression off.

[0250] Use of the transcriptional inhibitor fusion proteins of theinvention to inhibit transcription in eukaryotic cells also provideadvantages over the use of prokaryotic repressors alone (e.g., TetR,lacR) to inhibit transcription in eukaryotic cells. Since the inhibitorfusion proteins of the invention contain a eukaryotic transcriptionalsilencer domain, these fusion proteins should be more efficient atrepressing transcription in eukaryotic cells, and thus may potentiallyrequire lower intracellular concentrations for efficient repression withless liklihood of “leakiness”. Additionally, by insertion of tetOsequences into the regulatory region of an endogenous gene, thetranscriptional inhibitor fusion proteins of the invention can be usedto down-regulate constitutive and/or tissue-specific expression ofendogenous genes.

[0251] Furthermore, in contrast to various versions of the lac system(e.g., Labow et al. (1990) Mol. Cell. Biol. 10:3343-3356; Baim et al.(1991) Proc. Natl. Acad. Sci. USA 88:5072-5076), which are limited bythe negative properties of the inducing agent (IPTG) and/or by the needto increase the temperature in order to induce gene expression (whichmay elicit pleiotropic effects), the inducing agent used in the systemof the invention (Tc or an analogue thereof) has many advantageousproperties: 1) Tc and analogues thereof exhibit high affinity for TetRand low toxicity for eukaryotic cells, and thus can be used for geneinduction at concentrations that do not affect cell growth ormorphology; 2) Tc analogues which retain TetR binding but which havereduced antibiotic activity exist and can be used as inducing agents,thereby avoiding possible side effects from the antibiotic property ofTc; 3) the pharmacokinetic properties of Tc and Tc analogues enablerapid and efficient cellular uptake and penetration of physiologicalbarriers, such as the placenta or the blood-brain barrier; and 4) Tcanalogues with different induction capabilities permit modulation of thelevel of gene expression.

[0252] Thus, the invention provides an inducible regulatory system whichallows for rapid activation of gene transcription without cellulartoxicity and a range of induction indices. The increase in geneexpression upon induction typically is between 1000- and 2000-fold andcan be as high as about 20,000-fold. Alternatively, lower levels of geneinduction, e.g., 10-fold, can be achieved depending upon which inducingagent is used. This system can be utilized in a wide range ofapplications. These applications include gene therapy, large-scaleproduction of proteins in cultured cells or in transgenic farm animals,and the study of gene function, for example in relationship to cellulardevelopment and differentiation. Moreover, the novel transcription unitsof the invention allow for coordinate or independent regulation of theexpression of multiple genes utilizing the regulatory components of theinvention.

[0253] This invention is further illustrated by the following exampleswhich should not be construed as limiting. The contents of allreferences, patents and published patent applications cited throughoutthis application are hereby incorporated by reference.

EXAMPLE 1 Selection of a Mutated Tet Repressor and Construction of aTetracycline Inducible Transcriptional Activator

[0254] A “reverse” Tet repressor, which binds to its target DNA in thepresence rather than the absence of tetracycline, was generated bychemical mutagenesis and selection essentially as described in Hecht, B.et al. (1993) J. Bacteriology 175:1206-1210. Single-stranded DNA (codingand non-coding strands) encoding the wild-type Tn10-derived Tetrepressor was chemically mutagenized with sodium nitrite.Single-stranded DNAs (40 μg in 40 μl in Tris-EDTA buffer) were mixedwith 10 μl of 2.5 M sodium acetate (pH 4.3) and 50 μl of sodium nitrateranging between 0.25 M and 2 M and incubated for 45 to 60 minutes atroom temperature. After mutagenesis, the complementary strand wassynthesized using reverse transcriptase or by amplification using thepolymerase chain reaction with Taq DNA polymerase. Since the mutagenesisprocedure yields multiple mutations in the DNA, three fragments of thegene, of about 200 base pairs each, were individually subcloned into awt Tet repressor gene in a recombinant expression vector to replace thecorresponding portion of the wild-type gene. This created a pool ofmutated Tet repressor genes wherein each gene had mostly singlemutations in the 200 base pair mutagenized fragment of the gene.

[0255] The pool of mutated Tet repressors were screened in a geneticassay which positively selects for a functional interaction between aTet repressor and its cognate operator using E. coli. strainWH207(λWH25) (the construction of this strain is described in detail inWissmann, A. et al. (1991) Genetics 128:225-232). In this E. colistrain, tet operators direct the expression of divergently arrangedβ-galactoside (lacZ) and Lac repressor (lacI) genes and the lacregulatory region directs the expression of a galactokinase (galK) gene.Binding of Tet repressors to tet operators turns off transcription ofthe lacI and lacZ genes. The absence of Lac repressor allows forexpression of the galK gene, which enables the E. coli strain to usegalactose as a sole carbon source, which serves as one marker. The lacZ⁻phenotype serves as a second marker. Thus, bacteria containing Tetrepressors which bind to tet operators have a Gal⁺, lacZ⁻ phenotype.Bacteria containing wild-type Tet repressors have a Gal⁺, lacZ⁻phenotype in the absence of tetracycline. A mutated “reverse” Tetrepressor (rTetR) was selected based upon a Gal⁺, lacZ⁻ phenotype in thepresence of tetracycline.

[0256] The nucleotide and amino acid sequence of the rTetR mutant areshown in SEQ ID NOs: 1 (nucleotide positions 1-621) and 2 (amino acidpositions 1-207), respectively. Sequence analysis of the rTetR mutantshowed the following amino acid and nucleotide changes: aa (position)affected codon wild-type mutant wild-type mutant glu (71)  lys GAA AAAasp (95)  asn GAT AAT leu (101) ser TTA TCA gly (102) asp GGT GAT

[0257] Two additional mutations did not result in an amino acidexchange: aa (position) affected codon wild-type mutant wild-type mutantleu (41) leu TTG CTG arg (80) arg CGT CGC

[0258] To convert the rTetR mutant to a transcriptional activator, a 399base pair XbaI/Eco47III fragment encoding amino acids 3 to 135 of rTetR(i.e., encompassing the mutated region) was exchanged for thecorresponding restriction fragment of the expression vector pUHD15-1 tocreate pUHD17-1. In pUHD15-1, nucleotide sequences encoding wild-typeTetR are linked in frame to nucleotide sequences encoding the C-terminal130 amino acids of herpes simplex virus VP16. These transactivatorsequences are flanked upstream by a CMV promoter/enhancer and downstreamby an SV40 poly(A) site (the construction of pUHD15-1 is described inmore detail in U.S. Ser. No. 08/076,726 and Gossen, M. and Bujard, H.(1992) Proc. Natl. Acad. Sci. USA 89:5547-5551). Thus, in pUHD17-1,nucleotide sequences encoding the reverse TetR mutant are linked inframe to VP16 sequences to create a reverse Tc-controlled transactivator(referred to herein as tTA^(R)). The analogous exchange of the mutatedregion of rTetR for the wild-type region of TetR was performed withplasmid pUHD152-1, which is the same as pUHD15-1 except that itadditionally contains nucleotide sequences encoding a nuclearlocalization signal linked in-frame to the 5′ end of the nucleotidesequences encoding the Tet repressor. The amino acid sequence of thenuclear localization signal is MPKRPRP (SEQ ID NO: 5), which is linkedto the serine at amino acid position 2 of TetR. The resulting expressionvector encoding the reverse Tc-controlled transactivator including anuclear localization signal (referred to herein as ntTA^(R)) was namedpUHD172-1.

EXAMPLE 2 Tetracycline-Induced Stimulation of Transcription by tTA^(R)

[0259] Transient Transfection

[0260] The pUHD17-1 and pUHD172-1 expression vectors were transientlytransfected by a standard calcium phosphate method into HeLa cellstogether with a reporter plasmid, pUHC13-3, in which heptameric tetoperators are fused upstream of a minimal hCMV-promoter and a luciferasereporter gene (the reporter plasmid is described in detail in U.S. Ser.No. 08/076,726 and Gossen, M. and Bujard, H. (1992) Proc. Natl. Acad.Sci. USA 89:5547-5551). After incubation of the transfected cells at 37° C for 20 hours in the presence or absence of tetracyline (or ananalogue thereof), luciferase activity was assayed as follows: Cellsgrown to ˜80% confluency in 35 mm dishes in Eagle's minimal essentialmedium were washed with 2 ml of phosphate-buffered saline before theywere lysed in 25 mM Tris phosphate, pH 7.8/2 mM dithiothreitol/2 mMdiaminocyclohexanetetraacetic acid/10% glycerol/1% Triton-X-100 for 10minutes at room temperature. The lysate was scraped off the culturedishes and centrifuged for 10 seconds in an Eppendorf centrifuge. Next,aliquots (10 μl ) of the supernatant were mixed with 250 μl of 25 mMglycylglycine/15 mM MgSO4/5 mM ATP and assayed for luciferase activityin a Lumat LB9501 (Berthold, Wildbad, F. R. G.) using the integral mode(10 seconds). D-Luciferin (L6882, Sigma) was used at 0.5 mM. Thebackground signal measured in extracts of HeLa cells that did notcontain a luciferase gene was indistinguishable from the instrumentbackground (80-120 relative light units (rlu)/10 sec.). Protein contentof the lysate was determined according to Bradford (Bradford, M. M.(1976) Anal. Biochem. 72:248-254). Cells transfected with plasmidsencoding either tTA^(R) or ntTA^(R) showed an increased level ofluciferase activity in the presence of tetracyclines. This effect wasconsistently more pronounced when anhydrotetracycline (ATc) was usedinstead of tetracycline.

[0261] Stable Transfection

[0262] After this transient transfection analysis, expression vectorswere prepared for stable transfection of cells. A pSV2neo-derivedneomycin resistance cassette (described in Southern, P. J. and Berg, P.(1982) J. Mol. Appl. Genet. 1:327-341) was integrated into thetransactivator expression vectors pUHD17-1 and pUHD172-1, resulting inpUHD17-1neo and pUHD172-1neo, respectively. pUHD172-1neo, coding forntTA^(R), was stably integrated into HeLa cells by standard techniques.Ten G418-resistant cell clones were analyzed for their phenotype bytransient supertransfection with pUHC13-3 carrying the luciferase geneunder the control of a minimal CMV promoter and tet operators. Threeclones, HR4, HR5 and HR10, showed a strong increase of luciferaseactivity in the presence of ATc. From these clones, HR5 was selected forfurther experiments.

[0263] To create stable transfectants for both ntTA^(R) and a tetoperator-linked luciferase reporter gene, HR5 cells were cotransfectedwith pUH13-3 and pHMR272, which encodes for hygromycin resistance (seeBernhard, H-U. et al. (1985) Exp. Cell Res. 158:237-243), and hygromycinresistant clones were selected. In an analogous experiment, HR5 cellswere cotransfected with pUH13-7 and pHMR272. pUH13-7 contains a minimalpromoter sequence spanning position +19 to −37 of the HSVtk promoteradjacent to the heptameric tetO sequences, rather than a minimal CMVpromoter. From 21 hygromycin resistant clones, 10 showed inducibleluciferase activity upon addition of Tc or doxycyline (Dc) to theculture medium. Clones containing the luciferase reporter gene linked toa minimal CMV promoter are referred to as HR5-C, whereas thosecontaining the luciferase reporter gene linked to a minimal tk promoterare referred to HR5-T.

[0264] Six of the HR5 clones stably transfected with antTA^(R)-dependent reporter unit and previously shown to be responsiveto tetracyclines were grown in parallel in the absence or presence of 1μg/ml doxycycline. About 3×10⁴ cells were plated in each 35 mm dish (4dishes for each clone). After growth for 60 hours, cells were harvestedand the luciferase activity of the extracts (in relative light units(rlu)/μg extracted protein) was determined. As shown in Table 1, theabsolute expression levels of six clones demonstrate that activation ofluciferase gene expression over 3 orders of magnitude is achieved inseveral of the double stable cell lines containing the ntTA^(R)regulatory system.

[0265] It should be noted that even higher induction factors (e.g., ashigh as a 20,000-fold increase in expression) could be achieved if,instead of simply adding the inducing agent to the culture medium, thecells were washed prior to induction and then replated in fresh culturemedium containing the inducing agent. TABLE 1 Doxycycline-dependentluciferase activity of double stable luc+/HR5 cell clones LuciferaseActivity, rlu/μg protein Clone −Doxycycline +Doxycycline InductionFactor HR5-C6 65 54,911 845 62 69,525 1120 HR5-C11 100 165,671 1660 142179,651 1270 HR5-C14 43 44,493 1030 43 56,274 1310 HR5-T2 56 16,696 29840 16,416 410 HR5-T15 6.8 1838 270 6.5 1688 260 HR5-T19 4.8 1135 236 5.41285 237

[0266] Induction of Luciferase Activity by Different Tetracyclines

[0267] The ability of tetracycline and several different tetracyclineanalogues to induce luciferase expression in HR5-C11 cells was examined.HR5-C11 cells plated at a density of about 3×10⁴ cells/35 mm dish (˜80%confluency). After full attachment of the cells, the followingtetracyclines ere added to the cultures at a concentration of 1 μg/ml:tetracycline-HCl (Tc), oxytetracycline-HCl (OTc), chlorotetracycline(CTc), anhydrotetracycline-HCl (ATc) and doxycycline-HCl (Doxy). Thesecompounds are commercially available from Sigma Chemical Co., St. Louis,Mo., and were kept in aqueous solution at a concentration of 1 μg/ml.Cells grown in the absence of antibiotic (−) served as a control. After3 days, the cells were harvested and the luciferase activity and theprotein content of the extracts were determined. The results are shownin the bar graph of FIG. 1. Each bar in the figure (closed and hatched)represents the relative luciferase activity (normalized toward theamount of extracted protein) of a single culture dish. The mean of theluciferase activities obtained from the two plates grown withouttetracyclines was defined as 1. Tc, CTc and OTc showed modeststimulation of luciferase activity. By contrast, ATc and Doxy stimulatedluciferase activity approximately 1000 and 1500 fold, respectively.

[0268] Dose-response of Luciferase Activity to Doxycycline in HR5-C11Cells

[0269] The above-described experiment examining the induction ability ofdifferent tetracyclines revealed that doxycycline was the most potenteffector of the tetracyclines examined. Doxycycline was thereforeselected to quantitatively analyze its dose-response. HR5-C11 cells wereincubated with different concentrations of doxycycline and luciferaseactivity was measured. The data of three independent experiments areshown in FIG. 2. At less than 10 ng/ml in the culture medium,doxycycline is ineffective at inducing luciferase activity. However,when the concentration was raised above 10 ng/ml, an almost linearincrease in expression of luciferase was observed. Maximal activationwas achieved at 1 μg/ml. At concentrations above 3 μg/ml, doxycyclineshowed a slight growth-inhibitory effect on HeLa cells as determined ina MTT-assay.

[0270] Kinetics of Induction of the ntTA^(R) System

[0271] To examine the kinetics of doxycycline-induced ntTA^(R)-mediatedinduction of gene expression, the time course of induction of luciferaseactivity in HR5-C11 cells was monitored after addition of doxycycline tothe medium (final concentration 1 μg/ml). Cells were cultured in thepresence of doxycycline and after various time intervals, the cells wereharvested and luciferase activity was determined as described above. Asshown in FIG. 3, a 100-fold induction of luciferase activity wasobserved after 5.5 hours incubation with Doxy. Fully induced levels wereachieved in less than 24 hours of incubation with Doxy. Thus, theseresults indicate that induction of gene expression occurs rapidlyfollowing exposure of the cells to the inducing agent.

EXAMPLE 3 Coordinate Regulation of the Expression of Two NucleotideSequences by a Tc-Controlled Transcriptional Activator

[0272] A recombinant expression vector for coordinate, bidirectionaltranscription of two nucleotide sequences was constructed comprising, ina 5′ to 3′ direction: a luciferase gene, a first minimal promoter, seventet operator sequences, a second minimal promoter and a LacZ gene. Theconstruct is illustrated in FIG. 6. In this construct, the luciferaseand LacZ genes are oriented such that they are transcribed in oppositeorientations relative to the tet operator sequences, i.e., theluciferase gene is transcribed in a 5′ to 3′ direction from the bottomstrand of DNA, whereas the LacZ gene is transcribed in a 5′ to 3′direction from the top strand of DNA. The luciferase gene is followed byan SV40 polyadenylation signal, whereas the LacZ gene is followed by aβ-globin polyadenylation signal.

[0273] The construct was transfected into the HeLa cell line HtTA-1cells, which express a wild-type Tet repressor-VP16 fusion protein(referred to as tTA and described in Gossen, M. and Bujard, H. (1992)Proc. Natl. Acad. Sci. USA 89:5547-5551). The tTA fusion protein bindsto tet operator sequences in the absence of Tc (or analogue) but not inthe presence of Tc (or analogue). The construct was cotransfected intoHtTA-1 cells with a plasmid which confers hygromycin resistance andstably transfected clones were selected based upon their hygromycinresistant phenotype. Selected hygromycin resistant (Hygr^(r)) cloneswere examined for luciferase and β-galactosidase activity. Clonespositive for all three markers (Hygr^(r), luc⁺, β-gal⁺) were thenexamined for tetracycline-dependent coregulation of expression ofluciferase and β-galactosidase activity by cultureing the clones inincreasing amounts of tetracycline and measuring luciferase andβ-galactosidase activity. The results of such an experiment using cloneHt1316-8/50 are shown in FIG. 8. In the absence of tetracycline (inwhich case tTA can bind to tet operators and activate gene expression),both luciferase and β-galactosidase activity is detected. In thepresence of increasing amounts of tetracycline, luciferase andβ-galactosidase activity are coordinately and equivalentlydownregulated. This data demonstrates that expression of two genes canbe coordinately regulated by a tetracycline-controlled transactivator byoperatively linking the two genes to the same tet operator sequence(s).

EXAMPLE 4 Construction of a Tetracycline-Regulated TranscriptionalInhibitor Fusion Protein Comprising TetR and a Krueppel Silencer Domain

[0274] To contruct an expression vector encoding atetracycline-regulated transcriptional inhibitor of the invention (alsoreferred to as a tetacycline controlled silencer domain, or tSD), anucleic acid fragment encoding a transcriptional silencer domain isligated into an expression vector containing nucleotide sequencesencoding a wild-type or modified (i.e., mutated) TetR such that thesilencer domain coding sequences are ligated in-frame with the TetRcoding sequences. The plasmid pUHD141sma-1 contains nucleotide sequencesencoding a wild-type Tn10-derived Tet repressor (the nucleotide andamino acid sequences of which are shown in SEQ ID NOs: 16 and 17,respectively). In pUHD141sma-1, the TetR coding sequence is linked atits 5′ end to a CMV promoter and at its immediate 3′ end to a nucleotidesequence that creates a polylinker into which additional nucleic acidfragments can be introduced. The nucleotide sequence across thispolylinker region is: TCC CCG GGT AAC TAA GTA AGG ATC C (SEQ ID NO: 24)(wherein TCC CCG GGT ACC encode amino acid residues 205-208 of TetR,namely Ser-Gly-Ser-Asn). This polylinker region includes restrictionendonuclease sites for PspAI (CCC GGG) and BamHI (GGA TCC). Downstreamof the polylinker region, the plasmid contains an SV40-derivedpolyadenylation signal. The pUHD 141 sma-1 vector is illustratedschematically in FIG. 11.

[0275] To construct an expression vector encoding a fusion proteinbetween TetR and a transcriptional silencer domain from the DrosophilaKrueppel (Kr) protein, a nucleic acid fragment encoding a silencerdomain from Kr is amplified by the polymerase chain reaction (PCR) usingKr cDNA as a template. Oligonucleotide primers are designed whichamplify a nucleic acid fragment encoding the C-terminal 64 amino acidsof Kr (referred to as C64KR). This region corresponds to amino acidpositions 403-466 of the native protein. The nucleotide and amino acidsequences of C64KR are shown in SEQ ID NO: 20 and SEQ ID NO: 21,respectively. PCR primers are designed to include restrictionendonuclease sites such that the resultant amplified fragment containsrestriction endonuclease sites at its 5′ and 3′ ends. Restrictionendonuclease sites are chosen that are contained within the polylinkerof pUHD141sma-1 which allow in-frame, directional ligation of theamplified fragment into the polylinker site. For example, PCR primersare designed which incorporate a PspAI site (CCC GGG) at the 5′ end ofthe fragment encoding C64KR and a BamHI site at the 3′ end of thefragment. After a standard PCR reaction, the amplified fragment andpUHD141-sma1 are digested with PspAI and BamHI. The amplified fragmentis then ligated directionally into the polylinker site of pUHD141-sma1using standard ligation conditions to create the expression vectorpUHD141kr-1. Standard techniques are used to isolate the desired plasmidand confirm its construction. Construction of pUHD141kr-1 is illustratedschematically in FIG. 11.

[0276] The resultant pUHD141kr-1 expression vector contains nucleotidesequences encoding a fusion protein comprising amino acids 1-207 of thewild type TetR linked in-frame to amino acids 403466 of Kr (C64KR). Thenucleotide and amino acid sequences across the junction of the fusionprotein are as follows: AGT GGG TCC CCG GGT GAC ATG GAA (SEQ ID NO: 25)and Ser-Gly-Ser-Pro-Gly-Asp-Met-Glu (SEQ ID NO: 26). Ser-Gly-Sercorresponds to amino acids 205-207 of TetR, Pro-Gly are encoded by thepolylinker and Asp-Met-Glu correspond to the amino acids 403-405 ofC64KR.

[0277] Similarly, an expression vector encoding a fusion protein of amutated TetR (that binds to tetO only in the presence of Tc) and C64KRcan be constructed as described above using nucleotide sequencesencoding a mutant TetR (the nucleotide and amino acid sequences of whichare shown in SEQ ID NOs: 18 and 19, respectively) in place of the wildtype TetR sequences in pUHD141-sma1.

[0278] An expression vector encoding a TetR-Kr fusion protein (e.g.,pUHD141kr-1) is transiently or stably transfected into host cells asdescribed in Example 2 to express the TetR-Kr fusion protein in the hostcell. A reporter gene construct containing one or more tetO sequences, aminimal promoter and a reporter gene, such as luciferase, is alsotransfected into the cells as described in Example 2. (Reporter geneconstructs are described in further detail in U.S. Ser. No. 08/076,726and Gossen, M. and Bujard, H. (1992) Proc. Natl. Acad. Sci. USA89:5547-5551). Luciferase activity in the presence and absence ofincreasing concentrations of Tc or an analogue, e.g., doxycycline, ismeasured as described in Example 2. For the wild type TetR-Kr fusionprotein described above, the transcriptional inhibiting ability of thefusion protein is determined by comparing the amount of luciferaseactivity in the presence of doxycycline (no repression) to the amount ofluciferase activity in the absence of doxycycline (repression). Thetranscriptional inhibiting activity of the fusion protein can also betested using reporter gene constructs that exhibit higher basal levelsof expression (i.e., higher levels of expression in the presence ofdoxycycline) by using a reporter gene construct that contains additionalpositive regulatory elements (e.g., enhancer sequences).

EXAMPLE 5 Construction of a Tetracycline-Regulated TranscriptionalInhibitor Fusion Protein Comprising TetR and a v-erbA Silencer Domain

[0279] To construct an expression vector encoding a fusion proteinbetween TetR and a transcriptional silencer domain from the v-erbAoncogene product, a nucleic acid fragment encoding a silencer domainfrom v-erbA is ligated in-frame into pUHD141sma-1 as described inExample 4. A nucleic acid fragment encoding a v-erbA silencer domainsuitable for ligation into pUHD141sma-1 is amplified by the polymerasechain reaction (PCR) using a v-erbA cDNA as a template. Oligonucleotideprimers are designed which amplify a nucleic acid fragment encodingamino acids 364-635 of the native v-erbA protein. The nucleotide andamino acid sequences of this region of v-erbA are shown in SEQ ID NO: 22and SEQ ID NO: 23, respectively. As described in Example 4, PCR primersare designed such that the amplified v-erbA fragment containsrestriction endonuclease sites at its 5′ and 3′ ends, such as PspAI atthe 5′ end and BamHI at the 3′ end. After a standard PCR reaction, theamplified fragment and pUHD141-sma1 are digested with PspAI and BamHI.The amplified fragment is then ligated directionally into the polylinkersite of pUHD141-sma1 using standard ligation conditions to create theexpression vector pUHD 141 kr-1. Standard techniques can be used toisolate the desired plasmid and confirm its construction. Constructionof pUHD141erb-1 is illustrated schematically in FIG. 11.

[0280] The resultant pUHD141erb-1 expression vector contains nucleotidesequences encoding a fusion protein comprising a wild type TetR linkedin-frame to amino acids 364-635 of v-erbA. The nucleotide and amino acidsequences across the junction of the fusion protein are as follows: AGTGGG TCC CCG GGT CTG GAC GAC (SEQ ID NO: 27) andSer-Gly-Ser-Pro-Gly-Leu-Asp-Asp (SEQ ID NO: 28). Ser-Gly-Ser correspondsto amino acids 205-207 of TetR, Pro-Gly are encoded by the polylinkerand Leu-Asp-Asp correspond to amino acids 364-366 of the v-erbA silencerdomain.

[0281] As described in Example 4, an expression vector encoding a fusionprotein of a mutated TetR (that binds to tetO only in the presence ofTc) and a v-erbA silencer domain can be constructed as described aboveusing nucleotide sequences encoding a mutant TetR (the nucleotide andamino acid sequences of which are shown in SEQ ID NOs: 18 and 19,respectively) in place of the wild type TetR sequences in pUHD141-sma1.

[0282] Expression of the TetR-v-erbA fusion protein in host cells andassaying of the transcriptional inhibiting activity of the fusionprotein is as described in Example 4 for the TetR-Kr fusion protein.

EXAMPLE 6 Regulation of Gene Expression in Transgenic Animals by tTA^(R)

[0283] To examine the ability of tTA^(R) to regulate gene expression invivo, transgenic strains of mice were constructed which containedheterologous chromosomal insertions of either a tTA^(R) expressionconstruct or a reporter gene operably linked to tet operators. Singletransgenic strains containing either a tTA^(R) expression construct orthe tetO-linked reporter gene were then cross bred and double transgenicprogeny were identified. The double transgenic animals were thencharacterized as to the ability of tTA^(R), in a tetracycline dependentmanner, to regulate expression of the reporter gene. This exampledemonstrates that tTA^(R) effectively stimulates the expression of agene operably linked to tet operators in tissues of the animals in vivoupon administration of tetracycline (or analogue) to the animals,whereas expression of the tetO-linked gene remains at background levelsin the absence of tetracycline or an analogue. These results demonstratethat the tetracycline-controlled transcriptional regulatory systemdescribed herein functions effectively in animals, in addition to celllines in vitro.

[0284] Generation of Mice Transgenic for a P_(hCMV)-tTA^(R) ExpressionUnit

[0285] Mice expressing tTA protein were obtained by pronuclear injectioninto fertilized oocytes of a 2.7 kb XhoI-PfmI fragment excised fromplasmid pUHG17-1. This DNA fragment contained the tTA^(R) gene (shown inSEQ ID NO: 1) under the transcriptional control of the human CMV IEpromoter (position +75 to −675) together with a rabbit β-globinpolyadenylation site including an intron. The human CMV IE promoter is aconstitutive promoter that allows expression of the mutated tetR-VP16fusion protein in all cells where chromosomal integration of the DNAsequence encoding tTA^(R) has occurred. DNA was injected into fertilizedoocytes at a concentration of approximately 5 ng per μl by standardtechniques. Transgenic mice were generated from the injected fertilizedoocytes according to standard procedures. Transgenic founder mice wereanalyzed using polymerase chain reaction (PCR) and Southernhybridization to detect the presence of the tTA^(R) transgene inchromosomal DNA of the mice. Two transgenic mouse lines, CR3 and CR4were identified and crossbred with another transgenic mouse linecarrying a luciferase reporter gene under the control of tetO sequences.(described further below).

[0286] Generation of Mice Transgenic for the P_(hCMV*−1) LuciferaseReporter Unit

[0287] Mice carrying a P_(hCMV*−1) luc reporter gene expression unitwere generated by pronuclear injection into fertilized oocytes of a 3.1kb XhoI-EaeI fragment excised from plasmid pUHC13-3. This DNA-fragmentcontains the luciferase gene under transcriptional control of thetetracycline-responsive P_(hCMV*−1) promoter (SEQ ID NO: 8), togetherwith an SV40 t early polyadenylation site including an intron. DNA wasinjected into oocytes at a concentration of approximately 5 ng per μland transgenic mice were generated according to standard procedures.Transgenic founder mice were analyzed using Southern hybridization todetect the presence of the P_(hCMV*−1) luc transgene in chromosomal DNAof the mice. A mouse line transgenic for the tetO-linked luciferasereporter gene, L7, was crossbred with the tTA^(R) transgenic lines CR3and CR4 (described further below).

[0288] Generation of Mice Transgenic for the P_(hCMV*−1) luc andP_(hCMV)tTA^(R)

[0289] Having constructed single transgenic mice expressing tTA^(R) orcarrying P_(hCMV*−1) luc, double tansgenic mice carrying both the tTAexpression vector and the luciferase reporter-units were obtainedthrough cross breeding of heterozygous mice transgenic for one of thetwo transgenes. Double transgenic animals were identified by standardscreenings (e.g., PCR and/or Southern hybridization) to detect thepresence of both the tTA^(R) transgene and the P_(hCMV*−1) luc transgenein chromosomal DNA of the mice.

[0290] Induction and Analysis of Luciferase Activity in Tissue Samplesfrom Mice

[0291] For oral administration, tetracycline or its derivativedoxycycline were given in the drinking water at a concentration of 200μg per ml with 5% sucrose to hide the bitter taste of the antibiotics.For lactating mice, the concentration was 2 mg per ml with 10% sucroseto ensure a sufficient uptake via the milk by the young.

[0292] To analyze luciferase activity, mice were killed by cervicaldislocation and tissue samples were homogenized in 2 ml tubes containing500 μl lysis-buffer (25 mM Tris phosphate, pH 7.8/2 mM DTT/2 mM EDTA/10%glycerol/1% Triton X100) using a Ultra-Turrax. The homogenate was frozenin liquid nitrogen and centrifuged after thawing for 5 min at 15,000 g.2-20 μl of the supernatant were mixed with 250 μl luciferase assaybuffer (25 mM glycylglycine, pH 7.5/15 mM MgSO4/ 5 mM ATP) andluciferase activity was measured for 10 sec after the injection of 100μl of a 125 μM luciferin solution using Berthold Lumat LB 9501. Theprotein concentration of the homogenate was determined using Bradfordassay and luciferase activity was calculated as relative light units(rlu) per μg of total protein.

[0293] Results

[0294] Mice from 2 lines carrying the P_(hCMV)-tTA^(R) transgene (CR3and CR4) were mated with mice from line L7, transgenic for P_(hCMV*−1)luc. The L7 line shows a very low but detectable background ofluciferase activity in different organs that is probably due to positioneffects at the integration side. The background luciferase activity indifferent tissues of the L7 single transgenic mice is illustratedgraphically in FIG. 12, represented by the checked columns on theright-hand side for each tissue examined (each column represents theresults from one animals). The luciferase activity in different tissuesof the C3/L7 double transgenic mice in the absence of the tetracyclineanalogue doxycycline (i.e., uninduced conditions) is illustratedgraphically in FIG. 12, represented by the dark columns in the middlefor each tissue examined. The luciferase activity in different tissuesof the C3/L7 double transgenic mice in the presence of doxycycline(i.e., induced conditions) is illustrated graphically in FIG. 12,represented by the light columns on the left-hand side for each tissueexamined.

[0295] Luciferase activity was detectable in the seven tissues of thedouble transgenic mice examined: pancreas, kidney, stomach, muscle,thymus, heart and tongue. The tissue pattern of activated luciferaselevels (i.e., in the presence of doxycycline) in the double transgenicmice was similar to expression patterns of the hCMV IE promoter reportedin the literature. This is consistent with expression of the luciferasereporter gene being regulated by tTA^(R) (which is expressed in the miceunder the control of the hCMV IE promoter). The level of reporter geneinduction varied among the different tissues examined. Regulationfactors up to 100,000 fold (i.e., 5 orders of magnitude) were achieved,e.g. in the pancreas.

EXAMPLE 7 Combinatorial Regulatory Schemes Using Tetracycline-RegulatedFusion Proteins

[0296] In this example, the ability to use the varioustetracycline-regulated transcriptional activators and inhibitorsdescribed herein in combination, e.g., to regulate expression of twogene expression in a single cell using two different transactivators orone gene in a cell using both a transactivator and an inhibitor, wasinvestigated further. In a first series of experiments, various Tetrepressors having mutations within the DNA binding domain of the proteinwere examined for their ability to bind either a wild type tetO sequenceor various tetO variants to select for Tet repressors having specificityfor variant tetO sequences. In a second series of experiments, Tetrepressors having specificity for different variant tetO sequences wereconverted into either a tTA (which binds to its target tetO in theabsence of Tc) or a tTA^(R) (which binds to its target tetO in thepresence of Tc) and used to regulate gene expression in host cells. In athird series of experiments (described in Part C below), variouscombinations of different classes of Tet repressors were examined fortheir ability to heterodimerize to identify classes of Tet repressorswhich do not heterodimerize. Finally in Part D, below, the results fromthe three preceding analyses are combined to present preferred modelsystems for 1) regulating two tetO-linked genes in one cell using a tTAand a tTA^(R) having different variant tetO specificities and 2)regulating the expression of one tetO-linked gene using both aTc-regulated transcriptional activator and a Tc-regulatedtranscriptional inhibitor.

[0297] A. Tet Operator Binding Specificity of Tet Repressors Mutatedwithin the DNA Binding Domain

[0298] A series of amino acid substitutions were made in the Tn10 Tetrepressor within a region of the protein constituting the DNA bindingdomain (DBD) (i.e., the region of the protein which is thought tointeract with a target tetO sequence). Amino acid substitutions wereintroduced into TetR by standard mutagenesis of a DNA molecule encodingthe TetR. The ability of a DBD-mutated TetR to bind either a wild-type Bclass tetO or mutated variants thereof was then analyzed by introducingan expression vector encoding the DBD-mutated TetR into E. coli,together with a reporter gene comprising a β-galactosidase geneoperatively linked to either a wild-type tetO sequence (referred to as“4T”) or tetO sequences having a nucleotide substitution at either the 4or 6 position (“4C”, “6C”, “4A” or “4G”). The β-galactosidase activityin the cells was measured in the absence or presence of the TetRconstruct The ability of the TetR protein to bind to the tetO sequencewas determined by examining the % repression of P-galactosidase activityin the cell.

[0299] In a first series of experiments, a proline to glutamine mutationwas introduced at position 39 (“39PQ”), alone or together withadditional mutations at positions 37, 41 and/or 42. The results of theseanalyses are summarized below in Tables 2 and 3. In these tables,β-galactosidase activity in the absence of TetR is standardized as 100%.In the presence of a TetR that can bind the target tetO sequence linkedto the reporter gene, β-galactosidase activity is reduced (e.g., in thepresence of a wild-type TetR that can bind its target tetO sequence,β-galactosidase activity typically is less than 1%). In contrast, in thepresence of a DBD-mutated TetR that cannot bind the target tetO sequencelinked to the reporter gene, β-galactosidase activity remains near 100%.TABLE 2 tet Operator Tet Repressor 4C 4T (wt) 6C 4A 4G wt 57.3 ± 1.6 0.1± 0.1 47.1 ± 2.7 28 ± 0.7 88.3 ± 2.1 39PQ 46.7 ± 1.2 96.4 ± 0.1 88.4 ±4.5 97.3 ± 1.2 99.2 ± 4.2 37ES 9.3 ± 0.3 0.3 ± 0.1 35 ± 2.0 6 ± 0.1 64.2± 2.2 37ES39PQ 6.2 ± 0.5 92.5 ± 2 100.2 ± 2.3 95.8 ± 1.1 99.1 ± 3.937ES39PQ42YM 8.4 ± 0.3 96.7 ± 0.8 95.6 ± 1.1 98.8 ± 3 99.6 ± 3.437ES39PQ41LV 14.7 ± 1 95.1 ± 0.8 97.6 ± 2.8 102 ± 5.3 96.2 ± 3.137ES39PQ41LV42YM 0.2 ± 0.1 87.9 ± 0.7 96.1 ± 2.2 74.5 ± 3.4 88.2 ± 1.937ES39PQ41LI42YM 0.7 ± 0.2 87.7 ± 2.9 98.4 ± 1.2 79.4 ± 3.4 87.2 ± 0.637ES39PQ41LV42YS 99.9 ± 3.3 98.4 ± 2.1 98.8 ± 1.9 99.8 ± 1.6 100.1 ± 2.637ET 13.7 ± 0.6 0.2 ± 0.1 40.9 ± 2.9 8.1 ± 0.6 87.4 ± 2.5 37ET39PQ 4.8 ±0.2 93.5 ± 3.4 99.4 ± 2.2 102.3 ± 2.7 100.4 ± 1.7 37ET39PQ42YM 1.4 ± 093.8 ± 6.2 100.9 ± 3.9 103 ± 1.3 92.2 ± 0.6 37ET39PQ41LV 4.3 ± 0.2 92 ±0.9 100 ± 0.2 100.2 ± 4.2 97.9 ± 1.1 37ET39PQ41LV42YM 0.1 ± 0.1 95.8 ±0.1 104.2 ± 2.7 93.1 ± 1.4 91 ± 4.1 37EL 82 ± 0.4 0.4 ± 0.1 72.5 ± 4.884.8 ± 2.5 101.6 ± 1.9 37EL 39PQ 58.8 ± 0.5 95.6 ± 2.2 98.3 ± 4 98.9 ±2.7 102.4 ± 4.1 37EL39PQ42YM 8.2 ± 0.5 96.7 ± 0.3 99 ± 4.5 95.4 ± 2.5 97± 3 37EL39PQ41LV 73.9 ± 0.2 95.3 ± 0.4 98.2 ± 2.5 94.5 ± 0.9 98.1 ± 4.937EL39PQ41LV42YM 20.5 ± 0.1 92.3 ± 1.2 95.6 ± 2.9 96.1 ± 0.6 102.2 ± 4.837EL39PQ41LM42YM 0.7 ± 0.1 96.3 ± 2.2 98.2 ± 2.5 96.9 ± 1.2 102.6 ± 0.539PQ41LM 90.1 ± 2.1 97.2 ± 3.4 99.4 ± 3.4 96.5 ± 2.8 96.9 ± 2.2 39PQ42YM71.1 ± 1.8 95.6 ± 3.3 100.6 ± 1.6 97 ± 1.7 97.1 ± 8.2 39PQ41LM42YM 72.3± 0.7 98.6 ± 5.7 99.3 ± 0.4 93.5 ± 4.8 99.1 ± 4.5 41LM 94 ± 0.8 1.1 ± 095.4 ± 5 97.7 ± 1.1 103.1 ± 2 42YM 83.4 ± 0.2 20.2 ± 1 99.8 ± 3 100.3 ±2.6 100.4 ± 2.5 41LM42YM 88.5 ± 0.4 81.2 ± 4 103.4 ± 1.7 102.2 ± 4.799.2 ± 9.6 37EM 16.8 ± 1.4 1 ± 0.1 19.8 ± 0.5 37EM39PQ42YM 7.5 ± 0.9103.3 ± 7 99.3 ± 1.3 37EM39PQ41LV42YM 2.4 ± 0.3 102.9 ± 2.4 103.7 ± 337EV 7.9 ± 1.1 0.6 ± 0 40.3 ± 4.2 37EV39PQ 0.5 ± 0.1 93.2 ± 1.4 96.7 ±0.1 37EV39PQ42YM 0.2 ± 0.1 97.8 ± 0.8 95.5 ± 3.5 37EV39PQ41LV42YM 0.1 ±0 92.5 ± 1.8 97.4 ± 0.2 37EV39PQ41LT42YM 4.6 ± 0.9 93 ± 2.2 95.4 ± 0.237EF 81.3 ± 0.1 1.6 ± 0.2 6.4 ± 1.2 37EF39PQ 24.1 ± 3.8 96.6 ± 3.1 100 ±2.2 37EF39PQ42YM 10.4 ± 1 99.5 ± 4.7 101.6 ± 1.3 37EF39PQ41LV42YM 11.8 ±1.2 100 ± 8 100 ± 3.7 37EW 86.7 ± 1.4 2.8 ± 0.3 25.9 ± 2.3 37EW39PQ 51.8± 1.4 94.5 ± 2.6 100.2 ± 0.1 37EW39PQ42YM 40.7 ± 0 103.2 ± 3.4 103.1 ±3.9 37EW39PQ41LV42YM 40.8 ± 3 99.6 ± 8.6 101.2 ± 2 37EI 94.1 ± 1.8 3.1 ±0.9 100.1 ± 2.1 37EI39PQ 43.8 ± 1.6 94.2 ± 1.2 96.4 ± 0.2 37EI39PQ42YM2.4 ± 0.2 93.6 ± 1.8 96.6 ± 2.4 37EI39PQ41LV42YM 16.4 ± 1.2 99.2 ± 1.698.1 ± 0.3 37EH 61 ± 1.8 0.7 ± 0.1 2.2 ± 0.5 37EH39PQ 3.1 ± 0.2 76.5 ±2.7 99.2 ± 0.9 37EH39PQ42YM 15.7 ± 0.7 94.7 ± 0.8 98.6 ± 1.737EH39PQ41LV42YM 15.5 ± 1.8 99.7 ± 1.9 101.8 ± 4.4 37ER 7 ± 0.6 0.8 ±0.1 22.6 ± 2.3 37ER39PQ 0.3 ± 0.1 95.6 ± 0.8 95.8 ± 3.3 37ER39PQ41LV42YM0.1 ± 0.1 95.3 ± 1.9 95.6 ± 1.8

[0300] TABLE 3 tet Operator Tet Repressor 4C 6C 37ES39PQ41LI42YF 17.2 ±1.1 83.1 ± 0.7 37ES39PQ41LA42YF 31.3 ± 1   37ES39PQ41LP42YW 29.8 ± 0.537EA 8.5 ± 0.5 20.4 ± 0.3 37EA39PQ 1.5 ± 0.1 94.5 ± 5.1 37EA39PQ42YM 1.6± 0.3 92.6 ± 1.4 37EA39PQ41LV42YM 1.4 ± 0.2 98.3 ± 1.4 37EA39PQ41LM42YI47.7 ± 9.2 37EA39PQ41LA42YL 17.4 ± 0.5 37EA39PQ41LA42YW 24.9 ± 0.437EA39PQ41LW42YF 38.5 ± 0.5 37EV39PQ41LV42YC 8.3 ± 0.2 37EV39PQ41LI42YL26.9 ± 0.5 37EV39PQ41LM42YL 26.3 ± 0.3 37ER39PQ41LV42YL 22.7 ± 0.339ER39PQ41LM42YL 20.9 ± 0.8 37ER39PQ41LA42YW 35.9 ± 1.7 37ER39PQ41LI42YL21.8 ± 1.6 37EK 7.2 ± 0.4 20.8 ± 0.3 37EK39PQ 2.2 ± 0.5 101.3 ± 0.437EK39PQ42YF 10.8 ± 0.4 37EK39PQ41LT42YM 2 ± 0.1 103.4 ± 3  37EK39PQ41LW 10.2 ± 0.3 37EI39PQ41LF42YM 60.3 ± 0.5 37EQ 14.5 ± 0.1 13.2± 0.8 37EQ39PQ 2.7 ± 0.3 102.7 ± 1.6 37EQ39PQ42YM 6.1 ± 0.2 104.3 ± 2.337EQ39PQ41LT42YM 21.2 ± 0.6 37EG 89.8 ± 0.5 37EG39PQ 78.8 ± 0.637EG39PQ42YM 70.8 ± 1.5 37EG39PQ41LV42YM 64.9 ± 1.1 37EP 97.5 ± 7.237EP39PQ 87.1 ± 4.8 37EP39PQ42YM 64.6 ± 1.2 37ED 96.8 ± 6.1 37ED39PQ106.2 ± 1.9 37ED39PQ41LV42YM 88.2 ± 1.8 37EC 15.3 ± 0.3 37EC39PQ41LA42YM1.1 ± 0.1 37EY 88.8 ± 1.3 37EY39PQ41LA42YM 16.8 ± 1.4 37EN 102.2 ± 8.537EN39PQ 47.9 ± 0.5 39PQ42YM 71.1 ± 1.8 39PQ41LV42YM 49.4 ± 1.2

[0301] The results shown in Tables 2 and 3 demonstrate that theDBD-mutant TetR having only the 39PQ mutation cannot bind the wild-type(4T) tetO sequence (β-galactosidase activity remains at approximately96%, compared to 100% activity in the absence of the TetR). Furthermore,a series of mutants having, in addition to the 39PQ mutation, furthermutations at positions 37, 41 and/or 42 were identified that had thefollowing characteristics 1) an ability to efficiently bind to the “4C”tetO variant and 2) an inability to bind to the “6C” tetO variant. Suchmutants that could inhibit expression of a 4C tetO-linked reporter geneby at least 90% (i.e., β-galactosidase activity is less than or equal to10%) are indicated in bold type in Tables 2 and 3 above. These mutantsinclude: 37ES39PQ, 37ES39PQ42YM, 37ES39PQ41LV42YM, 37ES39PQ41LI42YM,37ET39PQ, 37ET39PQ42YM, 37ET39PQ41 LV, 37ET39PQ41LV42YM, 37EL39PQ42YM,37EL39PQ41 LM42YM, 37EM39PQ42YM, 37EM39PQ41LV42YM, 37EV39PQ,37EV39PQ42YM, 37EV39PQ41LV42YM, 37EV39PQ41LT42YM, 37EI39PQ42YM,37EH39PQ, 37ER39PQ, 37ER39PQ41LV42YM, 37EA39PQ, 37EA39PQ42YM,37EA39PQ41LV42YM, 37EK39PQ,37EK39PQ41LT42YM,37EQ39PQ and37EQ39PQ42YM.Even more preferred mutants that could inhibit expression of a 4CtetO-linked reporter gene by at least 98% (i.e., β-galactosidaseactivity is less than or equal to 2%) are indicated in bold type andunderlined in Tables 2 and 3 above. These mutants include:37ES39PQ41LV42YM, 37ES39PQ41 LI42YM, 37ET39PQ42YM, 37ET39PQ41 LV42YM,37EL39PQ41 LM42YM, 37EV39PQ, 37EV39PQ42YM, 37EV39PQ41LV42YM, 37ER39PQ,37ER39PQ41LV42YM, 37EA39PQ, 37EA39PQ42YM, 37EA39PQ41LV42YM and37EK39PQ41LT42YM.

[0302] In a second series of experiments, a preferred three-positionmutation identified above, 37EA39PQ42YM, was chosen for furtheranalysis. The binding specificities of one-, and two-position mutants(i.e., 37EA, 42YM, 37EA39PQ and 37EA42YM) were compared to thethree-position mutant (37EA39PQ42YM) using an expanded series of tetOvariants having nucleotide substitutions at either the 3,4, 5 or 6position. The results of these studies are summarize below in Tables 4and 5. TABLE 4 tetR Allele tet O2 EA37 EA37PQ39 EA37PQ39YM42 WT wt 0 ± 0< 67 ± 2  102 ± 0  0 ± 0 3C 6.1 ± 1.2 < 88 ± 4  < 104 ± 3  > 45 ± 1  3G54 ± 1  < 99 ± 3  − 114 ± 11  − 98 ± 2  3T 31 ± 2  < 99 ± 1  − 108 ±7  > 76 ± 3  4A 0.3 ± 0.1 < 74 ± 1  < 92 ± 3  > 6.0 ± 0.5 4C 0.5 ± 0.1 0± 0 0 ± 0 < 31 ± 2  4G 16 ± 1  < 83 ± 0  < 96 ± 1  > 69 ± 2  5C 2.6 ±0.6 < 16 ± 0  < 55 ± 0  > 1.6 ± 0.1 5G 46 ± 2  < 104 ± 2  − 100 ± 4  −94 ± 2  5T 0.4 ± 0.1 < 103 ± 3  − 109 ± 3  > 5.3 ± 1.3 6A 0 ± 0 < 100 ±5  − 105 ± 0  > 0.1 ± 0   6C 6 ± 0 < 102 ± 2  − 101 ± 1  > 18 ± 1 β-Galactosidase Activity in [%]

[0303] TABLE 5 tetR Allele tet O2 EA37 YM42 EA37YM42 WT wt 0 ± 0 4.4 ±1.4 0 ± 0 0 ± 0 3C 6.1 ± 1.2 98 ± 2  − 98 ± 5  > 45 ± 1  3G 54 ± 1  101± 2  − 106 ± 3  − 98 ± 2  3T 31 ± 2  102 ± 2  − 109 ± 2  > 76 ± 3  4A0.3 ± 0.1 68 ± 5  − 4.1 ± 0.1 < 6.0 ± 0.5 4C 0.5 ± 0.1 60 ± 4  > 0.2 ±0.1 < 31 ± 2  4G 16 ± 1  91 ± 1  − 32 ± 1  < 69 ± 2  5C 2.6 ± 0.6 48 ±3  > 1.2 ± 0   − 1.6 ± 0.1 5G 46 ± 2  101 ± 2  − 105 ± 2  − 94 ± 2  5T0.4 ± 0.1 80 ± 3  − 0.3 ± 0   < 5.3 ± 1.3 6A 0 ± 0 60 ± 0  − 21 ± 2  >0.1 ± 0   6C 6 ± 0 93 ± 1  − 48 ± 0  > 18 ± 1  β-Galactosidase Activityin [%]

[0304] The results of this analysis indicate that some of the one- andtwo-positions mutants, while retaining the ability to bind the 4Cvariant (like the three-position variant), have a broader bindingspecificity than the three-position variant. For example, 37EA42YMefficiently inhibits β-galactosidase activity of the 4C variant (0.2%activity), but also inhibits the activity of the 4A variant (4.1%activity), the 5C variant (1.2% activity) and the 5T variant (0.3%activity). In contrast, 37EA39PQ42YM efficiently inhibitsβ-galactosidase activity of the 4C variant (0% activity), but does notefficiently inhibit the activity of the 4A variant (92% activity), the5C variant (55% activity) or the ST variant (109% activity).

[0305] In a third series of experiments, a panel of TetR mutants havinga substitution at positions 43 and/or 44, alone or together withadditional mutations at positions 37, 40 and/or 41, were made and testedas described above. The results of these analyses are summarized belowin Table 6. TABLE 6 Operator Variant 6C wt 4C 6A 5G pWH1201 (Control)100% ± 6 100% ± 7 100% ± 6 100% ± 7 100% ± 3 pWH520 (WT TetR) 24% ± 10.2% ± 0.1 38% ± 3 0% ± 0.2 5% ± 1 43WR 68% ± 2 103% ± 3 95% ± 1 94% ± 390% ± 2 44HA 101% ± 2 91% ± 6 104% ± 5 96% ± 2 105% ± 3 43WR44HA 28% ±0.5 105% ± 7 102% ± 4 91% ± 2 90% ± 7 41LI43WR44HA 35% ± 1.5 101% ± 293% ± 3 97% ± 4 93% ± 1 44HV 97% ± 3 87% ± 2 107% ± 5 97% ± 2 106% ± 743WR44HV 36% ± 1.8 101% ± 3 95% ± 3 91% ± 4 97% ± 3 41LI43WR44HV 23% ± 196% ± 4 94% ± 4 83% ± 6 92% ± 4 40TR41LI43WR44HV 27% ± 1 105% ± 4 94% ±3 92% ± 5 91% ± 4 41L143WR44HT 25% ± 1 100% ± 3 97% ± 2 96% ± 2 94% ± 337ES 10% ± 0.3 0% ± 0.1 1.7% ± 0.2 0% ± 0 0% ± 0.1 37ES43WR44HN 3.8% ±0.3 80% ± 3 97% ± 5 104% ± 3 92% ± 3 37ES43WR44HV 5.5% ± 0.2 56% ± 2 94%± 3 80% ± 3 92% ± 0.5 41LV43WR44HV 87% ± 2 102% ± 2 93% ± 3 108% ± 3 95%± 3 37ES41LV43WR44HV 24% ± 0.7 77% ± 4 92% ± 3 103% ± 3 97% ± 1

[0306] The results shown in Table 6 demonstrate that DBD-mutant TetRshaving an ability to efficiently bind to the “6C” tetO variant but aninability to bind efficiently to the “4C” tetO variant can beidentified. Mutants that could inhibit expression of a 6C tetO-linkedreporter gene by at least 94% (i.e., β-galactosidase activity is lessthan or equal to 6%) are indicated in bold type in Table 6 above. Thetwo preferred mutants are 37ES43WR44HN (only 3.8% activity with the 6Cvariant but 97% activity with the 4C variant) and 37ES43WR44HV (only5.5% activity with the 6C variant but 94% activity with the 4C variant).The most preferred mutant is 37ES43WR44HN.

[0307] B. Use of tTA and tTA^(R) Transactivators with Different tetOBinding Specificities to Regulate Gene Expression in Cells

[0308] Tet repressor mutants having different DNA binding specificities,as described above in Part A, were used to construct expression vectorsencoding modified transactivator fusion proteins. One modified constructencoded a TetR having the DBD mutations 37EA39PQ42YM in an otherwisewild-type sequence, fused to VP16. This construct is referred to astTA₄. This construct binds to the 4C tetO variant but not the 6C tetOvariant and is regulated by Tc in the same manner as a wild-type TetR.Thus, this tTA₄ transactivator binds to 4C tetO sequences (but not 6CtetO sequences) in the absence, but not the presence, of Tc. Anothermodified construct encoded a TetR having the DBD mutations ES37WR43HN44,which confer the ability to bind the 6C tetO variant but not the 4C tetOvariant, and, additionally, the mutations 71EK95DN101LS102GD, whichconfer the “reverse” Tc-regulated phenotype, fused to VP16. Thisconstruct is referred to as tTA^(R) ₆. Thus, this tTA^(R) ₆ constructbinds to 6C tetO sequences (but not 4C tetO sequences) in the presence,but not the absence, of Tc.

[0309] Furthermore, the tetO-linked luciferase reporter gene constructPhCMV*−1 was modified to contain either the 4C or the 6C mutation. Thesemodified reporter gene constructs are referred to herein as tetO_(C4)and tetO_(C6), respectively.

[0310] To examine the ability of the tTA₄ and tTA^(R) ₆ expressionconstructs to regulate expression of the tetO_(C4) and tet_(C6) reportergene constructs within cells, various combinations of tansactivatorexpression constructs and reporter gene constructs were cotransfectedinto HeLa cells in transient transfection assays. The luciferaseactivity in cell extracts prepared from transfected cells culturedeither in the presence or absence of doxycycline was then examined.Representative results are illustrated graphically in FIG. 13.

[0311] The first pair of bars in FIG. 13 demonstrate that tTA₄efficiently regulates expression of tetO_(C4) (i.e., low luciferaseactivity in the presence of doxycycline but ˜1000-fold increase inluciferase activity in the absence of doxycycline). The second pair ofbars in FIG. 13 demonstrate that tTA^(R) ₆ efficiently regulatesexpression of tetO_(C6) (i.e., low luciferase activity in the absence ofdoxycycline but ˜100-fold increase in luciferase activity in thepresence of doxycycline). Finally, the third and fourth pairs of bars inFIG. 13 demonstrate that when both the tTA₄ and tTA^(R) ₆ constructs areintroduced into a single host cell their binding specificity for theirrespective operators, tetO_(C4) and tetO_(C6), is maintained (i.e., theydo not exhibit crossactivation of a non-target tetO sequence). Thus,when tTA₄ and tTA^(R) ₆ are both introduced into cells together with thetetO_(C4) reporter gene, expression of the reporter gene is regulated ina manner identical to when tTA₄ alone is introduced into the cell (i.e.,high luciferase activity only in the absence of doxycycline). Similarly,when tTA₄ and tTA^(R) ₆ are both introduced into cells together with thetetO_(C6) reporter gene, expression of the reporter gene is regulated ina manner identical to when tTA^(R) ₆ alone is introduced into the cell(i.e., high luciferase activity only in the presence of doxycycline).Additionally, when tTA₄ alone is cotransfected with tetO_(C6) or tTA^(R)₆ alone is cotransfected with tetO_(C4) no effect of the transactivatorsis observed. These various results demonstrate the strict DNA bindingspecificty of the tTA₄ and tTA^(R) ₆ fusion proteins and, moreover,demonstrate that the DNA binding specificity of an unfused DBD-mutatedTet repressor is maintained when the DBD-mutated Tet repressor isincorporated into a transcriptional activator fusion protein.

[0312] C. Dimerization Specificity of Different Classes of TetRepressors

[0313] Tet repressors can be placed into seven different classes calledA to E, G and H. Each repressor protein typically recognizes itsoperator sequences as a dimer. To establish an efficient combinationregulatory system, in which two (or more) Tc-regulated fusion proteinsare introduced into the same cell, it may be preferable to use classesof TetR that do not heterodimerize with each other (e.g., atransactivator fusion protein of one class and a transcriptionalinhibitor fusion protein of another class). To determine thedimerization specificity of TetRs of different classes, in vitrodimerization assays were performed. In this assay, two purified TetRwhich are distinguishable by their electrophoretic mobilities are mixedin a solution to avoid oxidation of the proteins and incubated for tenminutes at 50 ° C., followed by a 30 minute incubation at °4 C. Theprotein mixture is then analyzed by native polyacrylamide gelelectrophoresis.

[0314] A deletion mutant of the Tn10-encoded class B TetR (Δ26-53) wasused as one protein in the mixture. This deletion mutant has anincreased electrophoretic mobility compared to the full-length class B,C, D or E repressors tested. Thus, heterodimers between the deletionmutant and a full-length TetR can be detected by PAGE. When the class Bdeletion mutant was incubated with either a full-length class B TetR ora full-length class C tetR, heterodimers between the deletion mutant andthe full-length repressor were observed. In contrast, when the class Bdeletion mutant was incubated with either a full-length class D TetR ora full-length class E TetR, no heterodimer between the deletion mutantand the full-length repressor were observed.

[0315] To confirm these in vitro findings, in vivo transdominanceexperiments were performed. In these assays, a TetR expression constructwas introduced into an E. coli strain that carries a tetO-linkedβ-galactosidase reporter gene, either alone or together with anexpression construct for the class B TetR deletion mutant describedabove (Δ26-53), and β-galactosidase activity was measured. In thisassay, the level of β-galactosidase activity in the absence of any TetRconstructs was standardized as 100%. The presence of a full-length TetRconstruct alone results in decreased β-galactosidase activity. When afull-length TetR and the deletion construct are coexpressed in thecells, the deletion mutant cannot bind to tetO sequences itself but, ifit is able to heterodimerize with the full-length TetR, the deletionmutant will reduce the ability of the full-length TetR to repress β-galactivity. Thus, if the deletion mutant and the full-length TetRheterodimerize, β-gal activity is higher in the presence of the deletionmutant than in the absence of the deletion mutant. The results of theseexperiments are summarized below in Table 7. TABLE 7 Factor of Tet Levelof % β-galactosidase activity Trans- Repressor TetR Expression −Δ26-53+Δ26-53 dominance none — 100 100 — wt class B low 1.7 (±0.6) 37.3 (±3.5)22 wt class B high 1.2 (±0.2)  2.4 (±0.1) 2 wt class C low 84.5 (±1.3) 100.6 (±5.1)  1.2 wt class C high 6.4 (±0.0) 12.8 (±1.2) 2 wt class Dlow 62.2 (±3.5)  65.4 (±2.0) 1.1 wt class D high 1.4 (±0.0)  1.5 (±0.0)1.1 wt class E low 40.5 (±1.2)  41.1 (±3.8) 1.0 wt class E high 1.2(±0.1)  1.2 (±0.1) 1.0

[0316] The results shown in Table 7 confirm that the full-length class BTetR and the class B deletion mutant can heterodimerize (e.g., at lowlevels of TetR expression, β-galactosidase activity increases 22-fold inthe presence of the deletion mutant). Similarly, these experimentsindicate that the class B deletion mutant can heterodimerize with thefull-length class C TetR (e.g., at high levels of TetR expression,β-galactosidase activity increases 2-fold in the presence of thedeletion mutant). In contrast, no evidence of heterodimerization betweenthe class B deletion mutant and either the full-length class D or classE TetR was observed.

[0317] To confirm that results shown in Table 7 were not simply aspecific effect due to the particular deletion mutant used, similarexperiments were performed with two additional deletions mutants(TetR(B)Δ9-11 and TetR(B)Δ83-87). Similar results were observed with thetwo additional deletion mutants, confirming that the lack ofheterodimerization between class B TetR and either class D TetR or classE TetR was a general phenomenon that did not depend on which particulardeletion mutant was used.

[0318] Thus, based on in vitro and in vivo assays, a class B TetR doesnot efficiently heterodimerize with either a class D TetR or a class ETetR.

[0319] D. Model Regulatory Systems for Combination Regulation of Genes

[0320] The results discussed in the preceding sections can be combinedto create preferred model schemes for various combinatorial regulatorysystems. In the first combinatorial regulatory system, a single gene iscoordinately regulated by a reverse transactivator fusion protein and atranscriptional inhibitor fusion protein. This scheme is illustrated inFIG. 14, Model A. In this system, at least one tetO-4C variant sequenceis operatively linked to a minimal promoter (containg a “TATA” motif)and a gene of interest (“X”). Regulation of gene X in a cell ismediated, in part, by a “reverse activator” composed of a class B TetRhaving the DBD mutations 37EA39PQ42YM, which confer the ability to bindthe 4C variant, and, additionally, the mutations 71EK95DN101LS102GD,which confer the “reverse” Tc-regulated phenotype, fused to VP16. Thus,this reverse activator binds to tetO-4C in the presence of Tc to therebystimulate expression of the gene of interest. Regulation of the gene isalso mediated, in part, by a “silencer” composed of a class D TetRhaving the DBD mutations 37EAYM42, which confer the ability to bind the4C variant but also the ability to bind a broader spectrum of operatorsequences than the triple DBD mutation of the reverse transactivator,fused to a silencer domain, e.g., from v-erbA or Drosophila Krueppel.Thus, this silencer binds to tetO-4C in the absence of Tc to therebyinhibit expression of gene X. Based on the results in Part C above, theclass B TetR-containing reverse transactivator and the class DTetR-containing silencer will not heterodimerize. The combined use ofthe reverse transactivator and the silencer to regulate the gene ofinterest has the advantage that expression of the gene of interest ishighly repressed by the silencer in the absence of Tc but is efficientlystimulated by the reverse transactivator in the presence of Tc.

[0321] In the second combinatorial regulatory system, two genes in thesame cell are independently regulated by a tansactivator fusion proteinand a reverse transactivator fusion protein. This scheme is illustratedin FIG. 14, Model B. In this system, at least one tetO-4C variantsequence is operatively linked to a minimal promoter (“TATA”) and afirst gene of interest (“X”). Additionally, at least one tetO-6C variantsequence is operatively linked to a minimal promoter (“TATA”) and asecond gene of interest (“Y”). Regulation of gene X in a cell isaccomplished using an “activator” composed of a class B TetR having theDBD mutations 37EA39PQ42YM, which confer the ability to bind the 4Cvariant, in an otherwise wild-type sequence, fused to VP16. Thus, thisactivator binds to tetO-4C in the absence (but not the presence) of Tcto thereby stimulate expression of gene X. Regulation of gene Y in thecell is accomplished using a “reverse activator” composed of a class DTetR having the DBD mutations 37ESWR43HN44, which confer the ability tobind the 6C variant, and, additionally, the mutations71EK95DN101LS102GD, which confer the “reverse” Tc-regulated phenotype,fused to VP16. Thus, this reverse activator binds to tetO-6C in thepresence of Tc to thereby stimulate expression of gene Y. Based on theresults in Part C above, the class B TetR-containing transactivator andthe class D TetR-containing reverse transactivator will notheterodimerize. This combinatorial regulatory system thus allows forstimulation of one gene of interest in a cell in the absence of Tc andstimulation of another gene of interest in the same cell in the presenceof Tc.

[0322] EQUIVALENTS

[0323] Those skilled in the art will recognize, or be able to ascertainusing no more than routine experimentation, many equivalents to thespecific embodiments of the invention described herein. Such equivalentsare intended to be encompassed by the following claims.

1 28 1008 base pairs nucleic acid double linear DNA exon 1..1008 mRNA1..1008 misc. binding 1..207 misc. binding 208..335 CDS 1..1005 1 ATGTCT AGA TTA GAT AAA AGT AAA GTG ATT AAC AGC GCA TTA GAG CTG 48 Met SerArg Leu Asp Lys Ser Lys Val Ile Asn Ser Ala Leu Glu Leu 1 5 10 15 CTTAAT GAG GTC GGA ATC GAA GGT TTA ACA ACC CGT AAA CTC GCC CAG 96 Leu AsnGlu Val Gly Ile Glu Gly Leu Thr Thr Arg Lys Leu Ala Gln 20 25 30 AAG CTAGGT GTA GAG CAG CCT ACA CTG TAT TGG CAT GTA AAA AAT AAG 144 Lys Leu GlyVal Glu Gln Pro Thr Leu Tyr Trp His Val Lys Asn Lys 35 40 45 CGG GCT TTGCTC GAC GCC TTA GCC ATT GAG ATG TTA GAT AGG CAC CAT 192 Arg Ala Leu LeuAsp Ala Leu Ala Ile Glu Met Leu Asp Arg His His 50 55 60 ACT CAC TTT TGCCCT TTA AAA GGG GAA AGC TGG CAA GAT TTT TTA CGC 240 Thr His Phe Cys ProLeu Lys Gly Glu Ser Trp Gln Asp Phe Leu Arg 65 70 75 80 AAT AAG GCT AAAAGT TTT AGA TGT GCT TTA CTA AGT CAT CGC AAT GGA 288 Asn Lys Ala Lys SerPhe Arg Cys Ala Leu Leu Ser His Arg Asn Gly 85 90 95 GCA AAA GTA CAT TCAGAT ACA CGG CCT ACA GAA AAA CAG TAT GAA ACT 336 Ala Lys Val His Ser AspThr Arg Pro Thr Glu Lys Gln Tyr Glu Thr 100 105 110 CTC GAA AAT CAA TTAGCC TTT TTA TGC CAA CAA GGT TTT TCA CTA GAG 384 Leu Glu Asn Gln Leu AlaPhe Leu Cys Gln Gln Gly Phe Ser Leu Glu 115 120 125 AAT GCA TTA TAT GCACTC AGC GCT GTG GGG CAT TTT ACT TTA GGT TGC 432 Asn Ala Leu Tyr Ala LeuSer Ala Val Gly His Phe Thr Leu Gly Cys 130 135 140 GTA TTG GAA GAT CAAGAG CAT CAA GTC GCT AAA GAA GAA AGG GAA ACA 480 Val Leu Glu Asp Gln GluHis Gln Val Ala Lys Glu Glu Arg Glu Thr 145 150 155 160 CCT ACT ACT GATAGT ATG CCG CCA TTA TTA CGA CAA GCT ATC GAA TTA 528 Pro Thr Thr Asp SerMet Pro Pro Leu Leu Arg Gln Ala Ile Glu Leu 165 170 175 TTT GAT CAC CAAGGT GCA GAG CCA GCC TTC TTA TTC GGC CTT GAA TTG 576 Phe Asp His Gln GlyAla Glu Pro Ala Phe Leu Phe Gly Leu Glu Leu 180 185 190 ATC ATA TGC GGATTA GAA AAA CAA CTT AAA TGT GAA AGT GGG TCC GCG 624 Ile Ile Cys Gly LeuGlu Lys Gln Leu Lys Cys Glu Ser Gly Ser Ala 195 200 205 TAC AGC CGC GCGCGT ACG AAA AAC AAT TAC GGG TCT ACC ATC GAG GGC 672 Tyr Ser Arg Ala ArgThr Lys Asn Asn Tyr Gly Ser Thr Ile Glu Gly 210 215 220 CTG CTC GAT CTCCCG GAC GAC GAC GCC CCC GAA GAG GCG GGG CTG GCG 720 Leu Leu Asp Leu ProAsp Asp Asp Ala Pro Glu Glu Ala Gly Leu Ala 225 230 235 240 GCT CCG CGCCTG TCC TTT CTC CCC GCG GGA CAC ACG CGC AGA CTG TCG 768 Ala Pro Arg LeuSer Phe Leu Pro Ala Gly His Thr Arg Arg Leu Ser 245 250 255 ACG GCC CCCCCG ACC GAT GTC AGC CTG GGG GAC GAG CTC CAC TTA GAC 816 Thr Ala Pro ProThr Asp Val Ser Leu Gly Asp Glu Leu His Leu Asp 260 265 270 GGC GAG GACGTG GCG ATG GCG CAT GCC GAC GCG CTA GAC GAT TTC GAT 864 Gly Glu Asp ValAla Met Ala His Ala Asp Ala Leu Asp Asp Phe Asp 275 280 285 CTG GAC ATGTTG GGG GAC GGG GAT TCC CCG GGT CCG GGA TTT ACC CCC 912 Leu Asp Met LeuGly Asp Gly Asp Ser Pro Gly Pro Gly Phe Thr Pro 290 295 300 CAC GAC TCCGCC CCC TAC GGC GCT CTG GAT ATG GCC GAC TTC GAG TTT 960 His Asp Ser AlaPro Tyr Gly Ala Leu Asp Met Ala Asp Phe Glu Phe 305 310 315 320 GAG CAGATG TTT ACC GAT CCC CTT GGA ATT GAC GAG TAC GGT GGG TAG 1008 Glu Gln MetPhe Thr Asp Pro Leu Gly Ile Asp Glu Tyr Gly Gly 325 330 335 335 aminoacids amino acid linear protein 2 Met Ser Arg Leu Asp Lys Ser Lys ValIle Asn Ser Ala Leu Glu Leu 1 5 10 15 Leu Asn Glu Val Gly Ile Glu GlyLeu Thr Thr Arg Lys Leu Ala Gln 20 25 30 Lys Leu Gly Val Glu Gln Pro ThrLeu Tyr Trp His Val Lys Asn Lys 35 40 45 Arg Ala Leu Leu Asp Ala Leu AlaIle Glu Met Leu Asp Arg His His 50 55 60 Thr His Phe Cys Pro Leu Lys GlyGlu Ser Trp Gln Asp Phe Leu Arg 65 70 75 80 Asn Lys Ala Lys Ser Phe ArgCys Ala Leu Leu Ser His Arg Asn Gly 85 90 95 Ala Lys Val His Ser Asp ThrArg Pro Thr Glu Lys Gln Tyr Glu Thr 100 105 110 Leu Glu Asn Gln Leu AlaPhe Leu Cys Gln Gln Gly Phe Ser Leu Glu 115 120 125 Asn Ala Leu Tyr AlaLeu Ser Ala Val Gly His Phe Thr Leu Gly Cys 130 135 140 Val Leu Glu AspGln Glu His Gln Val Ala Lys Glu Glu Arg Glu Thr 145 150 155 160 Pro ThrThr Asp Ser Met Pro Pro Leu Leu Arg Gln Ala Ile Glu Leu 165 170 175 PheAsp His Gln Gly Ala Glu Pro Ala Phe Leu Phe Gly Leu Glu Leu 180 185 190Ile Ile Cys Gly Leu Glu Lys Gln Leu Lys Cys Glu Ser Gly Ser Ala 195 200205 Tyr Ser Arg Ala Arg Thr Lys Asn Asn Tyr Gly Ser Thr Ile Glu Gly 210215 220 Leu Leu Asp Leu Pro Asp Asp Asp Ala Pro Glu Glu Ala Gly Leu Ala225 230 235 240 Ala Pro Arg Leu Ser Phe Leu Pro Ala Gly His Thr Arg ArgLeu Ser 245 250 255 Thr Ala Pro Pro Thr Asp Val Ser Leu Gly Asp Glu LeuHis Leu Asp 260 265 270 Gly Glu Asp Val Ala Met Ala His Ala Asp Ala LeuAsp Asp Phe Asp 275 280 285 Leu Asp Met Leu Gly Asp Gly Asp Ser Pro GlyPro Gly Phe Thr Pro 290 295 300 His Asp Ser Ala Pro Tyr Gly Ala Leu AspMet Ala Asp Phe Glu Phe 305 310 315 320 Glu Gln Met Phe Thr Asp Pro LeuGly Ile Asp Glu Tyr Gly Gly 325 330 335 33 base pairs nucleic aciddouble linear DNA 3 GAC GCG CTA GAC GAT TTC GAT CTG GAC ATG TTG 33 AspAla Leu Asp Asp Phe Asp Leu Asp Met Leu 1 5 10 11 amino acids amino acidlinear peptide internal 4 Asp Ala Leu Asp Asp Phe Asp Leu Asp Met Leu 15 10 7 amino acids amino acid linear peptide internal 5 Met Pro Lys ArgPro Arg Pro 1 5 569 base pairs nucleic acid double linear DNA 6GAATTCGGGG CCGCGGAGGC TGGATCGGTC CCGGTGTCTT CTATGGAGGT CAAAACAGCG 60TGGATGGCGT CTCCAGGCGA TCTGACGGTT CACTAAACGA GCTCTGCTTA TATAGGTCGA 120GTTTACCACT CCCTATCAGT GATAGAGAAA AGTGAAAGTC GAGTTTACCA CTCCCTATCA 180GTGATAGAGA AAAGTGAAAG TCGAGTTTAC CACTCCCTAT CAGTGATAGA GAAAAGTGAA 240AGTCGAGTTT ACCACTCCCT ACCAGTGATA GAGAAAAGTG AAAGTCGAGT TTACCACTCC 300CTATCAGTGA TAGAGAAAAG TGAAAGTCGA GTTTACCACT CCCTATCAGT GATAGAGAAA 360AGTGAAAGTC GAGTTTACCA CTCCCTATCA GTGATAGAGA AAAGTGAAAG TCGAGCTCGG 420TACCCGGGTC GAGTAGGCGT GTACGGTGGG AGGCCTATAT AAGCAGAGCT CGTTTAGTGA 480ACCGTCAGAT CGCCTGGAGA CGCCATCCAC GCTGTTTTGA CCTCCATAGA AGACACCGGG 540ACCGATCCAG CCTCCGCGGC CCCGAATTC 569 520 base pairs nucleic acid doublelinear DNA 7 AGATCTGCAG GGTCGCTCGG TGTTCGAGGC CACACGCGTC ACCTTAATATGCGAAGTGGA 60 CCGGATCTCG AGTTTACCAC TCCCTATCAG TGATAGAGAA AAGTGAAAGTCGAGTTTACC 120 ACTCCCTATC AGTGATAGAG AAAAGTGAAA GTCGAGTTTA CCACTCCCTATCAGTGATAG 180 AGAAAAGTGA AAGTCGAGTT TACCACTCCC TATCAGTGAT AGAGAAAAGTGAAAGTCGAG 240 TTTACCACTC CCTATCAGTG ATAGAGAAAA GTGAAAGTCG AGTTTACCACTCCCTATCAG 300 TGATAGAGAA AAGTGAAAGT CGAGTTTACC ACTCCCTATC AGTGATAGAGAAAAGTGAAA 360 GTCGAGCTCG GTACCCGGGT CGAGTAGGCG TGTACGGTGG GAGGCCTATATAAGCAGAGC 420 TCGTTTAGTG AACCGTCAGA TCGCCTGGAG ACGCCATCCA CGCTGTTTTGACCTCCATAG 480 AAGACACCGG GACCGATCCA GCCTCCGCGG CCCCGAATTC 520 450 basepairs nucleic acid double linear DNA (genomic) Human cytomegalovirusK12, Towne mRNA 382..450 8 GAATTCCTCG AGTTTACCAC TCCCTATCAG TGATAGAGAAAAGTGAAAGT CGAGTTTACC 60 ACTCCCTATC AGTGATAGAG AAAAGTGAAA GTCGAGTTTACCACTCCCTA TCAGTGATAG 120 AGAAAAGTGA AAGTCGAGTT TACCACTCCC TATCAGTGATAGAGAAAAGT GAAAGTCGAG 180 TTTACCACTC CCTATCAGTG ATAGAGAAAA GTGAAAGTCGAGTTTACCAC TCCCTATCAG 240 TGATAGAGAA AAGTGAAAGT CGAGTTTACC ACTCCCTATCAGTGATAGAG AAAAGTGAAA 300 GTCGAGCTCG GTACCCGGGT CGAGTAGGCG TGTACGGTGGGAGGCCTATA TAAGCAGAGC 360 TCGTTTAGTG AACCGTCAGA TCGCCTGGAG ACGCCATCCACGCTGTTTTG ACCTCCATAG 420 AAGACACCGG GACCGATCCA GCCTCCGCGG 450 450 basepairs nucleic acid double linear DNA (genomic) Human cytomegalovirusTowne mRNA 382..450 9 GAATTCCTCG ACCCGGGTAC CGAGCTCGAC TTTCACTTTTCTCTATCACT GATAGGGAGT 60 GGTAAACTCG ACTTTCACTT TTCTCTATCA CTGATAGGGAGTGGTAAACT CGACTTTCAC 120 TTTTCTCTAT CACTGATAGG GAGTGGTAAA CTCGACTTTCACTTTTCTCT ATCACTGATA 180 GGGAGTGGTA AACTCGACTT TCACTTTTCT CTATCACTGATAGGGAGTGG TAAACTCGAC 240 TTTCACTTTT CTCTATCACT GATAGGGAGT GGTAAACTCGACTTTCACTT TTCTCTATCA 300 CTGATAGGGA GTGGTAAACT CGAGTAGGCG TGTACGGTGGGAGGCCTATA TAAGCAGAGC 360 TCGTTTAGTG AACCGTCAGA TCGCCTGGAG ACGCCATCCACGCTGTTTTG ACCTCCATAG 420 AAGACACCGG GACCGATCCA GCCTCCGCGG 450 398 basepairs nucleic acid double linear DNA (genomic) Herpes Simplex Virus KOS10 GAGCTCGACT TTCACTTTTC TCTATCACTG ATAGGGAGTG GTAAACTCGA CTTTCACTTT 60TCTCTATCAC TGATAGGGAG TGGTAAACTC GACTTTCACT TTTCTCTATC ACTGATAGGG 120AGTGGTAAAC TCGACTTTCA CTTTTCTCTA TCACTGATAG GGAGTGGTAA ACTCGACTTT 180CACTTTTCTC TATCACTGAT AGGGAGTGGT AAACTCGACT TTCACTTTTC TCTATCACTG 240ATAGGGAGTG GTAAACTCGA CTTTCACTTT TCTCTATCAC TGATAGGGAG TGGTAAACTC 300GAGATCCGGC GAATTCGAAC ACGCAGATGC AGTCGGGGCG GCGCGGTCCG AGGTCCACTT 360CGCATATTAA GGTGACGCGT GTGGCCTCGA ACACCGAG 398 38 base pairs nucleic aciddouble linear DNA 11 ACTTTATCAC TGATAAACAA ACTTATCAGT GATAAAGA 38 38base pairs nucleic acid double linear DNA 12 ACTCTATCAT TGATAGAGTTCCCTATCAGT GATAGAGA 38 38 base pairs nucleic acid double linear DNA 13AGCTTATCAT CGATAAGCTA GTTTATCACA GTTAAATT 38 38 base pairs nucleic aciddouble linear DNA 14 ACTCTATCAT TGATAGGGAA CTCTATCAAT GATAGGGA 38 38base pairs nucleic acid double linear DNA 15 AATCTATCAC TGATAGAGTACCCTATCATC GATAGAGA 38 621 base pairs nucleic acid double linear cDNA 16ATG TCT AGA TTA GAT AAA AGT AAA GTG ATT AAC AGC GCA TTA GAG CTG 48 MetSer Arg Leu Asp Lys Ser Lys Val Ile Asn Ser Ala Leu Glu Leu 1 5 10 15CTT AAT GAG GTC GGA ATC GAA GGT TTA ACA ACC CGT AAA CTC GCC CAG 96 LeuAsn Glu Val Gly Ile Glu Gly Leu Thr Thr Arg Lys Leu Ala Gln 20 25 30 AAGCTA GGT GTA GAG CAG CCT ACA TTG TAT TGG CAT GTA AAA AAT AAG 144 Lys LeuGly Val Glu Gln Pro Thr Leu Tyr Trp His Val Lys Asn Lys 35 40 45 CGG GCTTTG CTC GAC GCC TTA GCC ATT GAG ATG TTA GAT AGG CAC CAT 192 Arg Ala LeuLeu Asp Ala Leu Ala Ile Glu Met Leu Asp Arg His His 50 55 60 ACT CAC TTTTGC CCT TTA GAA GGG GAA AGC TGG CAA GAT TTT TTA CGT 240 Thr His Phe CysPro Leu Glu Gly Glu Ser Trp Gln Asp Phe Leu Arg 65 70 75 80 AAT AAG GCTAAA AGT TTT AGA TGT GCT TTA CTA AGT CAT CGC GAT GGA 288 Asn Lys Ala LysSer Phe Arg Cys Ala Leu Leu Ser His Arg Asp Gly 85 90 95 GCA AAA GTA CATTTA GGT ACA CGG CCT ACA GAA AAA CAG TAT GAA ACT 336 Ala Lys Val His LeuGly Thr Arg Pro Thr Glu Lys Gln Tyr Glu Thr 100 105 110 CTC GAA AAT CAATTA GCC TTT TTA TGC CAA CAA GGT TTT TCA CTA GAG 384 Leu Glu Asn Gln LeuAla Phe Leu Cys Gln Gln Gly Phe Ser Leu Glu 115 120 125 AAT GCA TTA TATGCA CTC AGC GCT GTG GGG CAT TTT ACT TTA GGT TGC 432 Asn Ala Leu Tyr AlaLeu Ser Ala Val Gly His Phe Thr Leu Gly Cys 130 135 140 GTA TTG GAA GATCAA GAG CAT CAA GTC GCT AAA GAA GAA AGG GAA ACA 480 Val Leu Glu Asp GlnGlu His Gln Val Ala Lys Glu Glu Arg Glu Thr 145 150 155 160 CCT ACT ACTGAT AGT ATG CCG CCA TTA TTA CGA CAA GCT ATC GAA TTA 528 Pro Thr Thr AspSer Met Pro Pro Leu Leu Arg Gln Ala Ile Glu Leu 165 170 175 TTT GAT CACCAA GGT GCA GAG CCA GCC TTC TTA TTC GGC CTT GAA TTG 576 Phe Asp His GlnGly Ala Glu Pro Ala Phe Leu Phe Gly Leu Glu Leu 180 185 190 ATC ATA TGCGGA TTA GAA AAA CAA CTT AAA TGT GAA AGT GGG TCC 621 Ile Ile Cys Gly LeuGlu Lys Gln Leu Lys Cys Glu Ser Gly Ser 195 200 205 207 amino acidsamino acid linear protein 17 Met Ser Arg Leu Asp Lys Ser Lys Val Ile AsnSer Ala Leu Glu Leu 1 5 10 15 Leu Asn Glu Val Gly Ile Glu Gly Leu ThrThr Arg Lys Leu Ala Gln 20 25 30 Lys Leu Gly Val Glu Gln Pro Thr Leu TyrTrp His Val Lys Asn Lys 35 40 45 Arg Ala Leu Leu Asp Ala Leu Ala Ile GluMet Leu Asp Arg His His 50 55 60 Thr His Phe Cys Pro Leu Glu Gly Glu SerTrp Gln Asp Phe Leu Arg 65 70 75 80 Asn Lys Ala Lys Ser Phe Arg Cys AlaLeu Leu Ser His Arg Asp Gly 85 90 95 Ala Lys Val His Leu Gly Thr Arg ProThr Glu Lys Gln Tyr Glu Thr 100 105 110 Leu Glu Asn Gln Leu Ala Phe LeuCys Gln Gln Gly Phe Ser Leu Glu 115 120 125 Asn Ala Leu Tyr Ala Leu SerAla Val Gly His Phe Thr Leu Gly Cys 130 135 140 Val Leu Glu Asp Gln GluHis Gln Val Ala Lys Glu Glu Arg Glu Thr 145 150 155 160 Pro Thr Thr AspSer Met Pro Pro Leu Leu Arg Gln Ala Ile Glu Leu 165 170 175 Phe Asp HisGln Gly Ala Glu Pro Ala Phe Leu Phe Gly Leu Glu Leu 180 185 190 Ile IleCys Gly Leu Glu Lys Gln Leu Lys Cys Glu Ser Gly Ser 195 200 205 621 basepairs nucleic acid double linear cDNA 18 ATG TCT AGA TTA GAT AAA AGT AAAGTG ATT AAC AGC GCA TTA GAG CTG 48 Met Ser Arg Leu Asp Lys Ser Lys ValIle Asn Ser Ala Leu Glu Leu 1 5 10 15 CTT AAT GAG GTC GGA ATC GAA GGTTTA ACA ACC CGT AAA CTC GCC CAG 96 Leu Asn Glu Val Gly Ile Glu Gly LeuThr Thr Arg Lys Leu Ala Gln 20 25 30 AAG CTA GGT GTA GAG CAG CCT ACA CTGTAT TGG CAT GTA AAA AAT AAG 144 Lys Leu Gly Val Glu Gln Pro Thr Leu TyrTrp His Val Lys Asn Lys 35 40 45 CGG GCT TTG CTC GAC GCC TTA GCC ATT GAGATG TTA GAT AGG CAC CAT 192 Arg Ala Leu Leu Asp Ala Leu Ala Ile Glu MetLeu Asp Arg His His 50 55 60 ACT CAC TTT TGC CCT TTA AAA GGG GAA AGC TGGCAA GAT TTT TTA CGC 240 Thr His Phe Cys Pro Leu Lys Gly Glu Ser Trp GlnAsp Phe Leu Arg 65 70 75 80 AAT AAG GCT AAA AGT TTT AGA TGT GCT TTA CTAAGT CAT CGC AAT GGA 288 Asn Lys Ala Lys Ser Phe Arg Cys Ala Leu Leu SerHis Arg Asn Gly 85 90 95 GCA AAA GTA CAT TCA GAT ACA CGG CCT ACA GAA AAACAG TAT GAA ACT 336 Ala Lys Val His Ser Asp Thr Arg Pro Thr Glu Lys GlnTyr Glu Thr 100 105 110 CTC GAA AAT CAA TTA GCC TTT TTA TGC CAA CAA GGTTTT TCA CTA GAG 384 Leu Glu Asn Gln Leu Ala Phe Leu Cys Gln Gln Gly PheSer Leu Glu 115 120 125 AAT GCA TTA TAT GCA CTC AGC GCT GTG GGG CAT TTTACT TTA GGT TGC 432 Asn Ala Leu Tyr Ala Leu Ser Ala Val Gly His Phe ThrLeu Gly Cys 130 135 140 GTA TTG GAA GAT CAA GAG CAT CAA GTC GCT AAA GAAGAA AGG GAA ACA 480 Val Leu Glu Asp Gln Glu His Gln Val Ala Lys Glu GluArg Glu Thr 145 150 155 160 CCT ACT ACT GAT AGT ATG CCG CCA TTA TTA CGACAA GCT ATC GAA TTA 528 Pro Thr Thr Asp Ser Met Pro Pro Leu Leu Arg GlnAla Ile Glu Leu 165 170 175 TTT GAT CAC CAA GGT GCA GAG CCA GCC TTC TTATTC GGC CTT GAA TTG 576 Phe Asp His Gln Gly Ala Glu Pro Ala Phe Leu PheGly Leu Glu Leu 180 185 190 ATC ATA TGC GGA TTA GAA AAA CAA CTT AAA TGTGAA AGT GGG TCC 621 Ile Ile Cys Gly Leu Glu Lys Gln Leu Lys Cys Glu SerGly Ser 195 200 205 207 amino acids amino acid linear protein 19 Met SerArg Leu Asp Lys Ser Lys Val Ile Asn Ser Ala Leu Glu Leu 1 5 10 15 LeuAsn Glu Val Gly Ile Glu Gly Leu Thr Thr Arg Lys Leu Ala Gln 20 25 30 LysLeu Gly Val Glu Gln Pro Thr Leu Tyr Trp His Val Lys Asn Lys 35 40 45 ArgAla Leu Leu Asp Ala Leu Ala Ile Glu Met Leu Asp Arg His His 50 55 60 ThrHis Phe Cys Pro Leu Lys Gly Glu Ser Trp Gln Asp Phe Leu Arg 65 70 75 80Asn Lys Ala Lys Ser Phe Arg Cys Ala Leu Leu Ser His Arg Asn Gly 85 90 95Ala Lys Val His Ser Asp Thr Arg Pro Thr Glu Lys Gln Tyr Glu Thr 100 105110 Leu Glu Asn Gln Leu Ala Phe Leu Cys Gln Gln Gly Phe Ser Leu Glu 115120 125 Asn Ala Leu Tyr Ala Leu Ser Ala Val Gly His Phe Thr Leu Gly Cys130 135 140 Val Leu Glu Asp Gln Glu His Gln Val Ala Lys Glu Glu Arg GluThr 145 150 155 160 Pro Thr Thr Asp Ser Met Pro Pro Leu Leu Arg Gln AlaIle Glu Leu 165 170 175 Phe Asp His Gln Gly Ala Glu Pro Ala Phe Leu PheGly Leu Glu Leu 180 185 190 Ile Ile Cys Gly Leu Glu Lys Gln Leu Lys CysGlu Ser Gly Ser 195 200 205 192 base pairs nucleic acid double linearcDNA 20 GAC ATG GAA AAA GCG ACA CCG GAG ACG ATG GTC CAT TGG ATT TGT CTG48 Asp Met Glu Lys Ala Thr Pro Glu Thr Met Val His Trp Ile Cys Leu 1 510 15 AAG ATG GAG CCA GCT CTG TGG ATG GCC ATT ACA GCA ACA TCG CAC GGC 96Lys Met Glu Pro Ala Leu Trp Met Ala Ile Thr Ala Thr Ser His Gly 20 25 30GCA AGG CAC AGG ACA TTC GTC GGG TTT TCC GGC TGC CTC CAC CGC AAA 144 AlaArg His Arg Thr Phe Val Gly Phe Ser Gly Cys Leu His Arg Lys 35 40 45 TCCCTC ACG TAC CCA GTG ATA TGC CTG AGC AAA CCG AGC CAG AGG ATT 192 Ser LeuThr Tyr Pro Val Ile Cys Leu Ser Lys Pro Ser Gln Arg Ile 50 55 60 64amino acids amino acid linear protein 21 Asp Met Glu Lys Ala Thr Pro GluThr Met Val His Trp Ile Cys Leu 1 5 10 15 Lys Met Glu Pro Ala Leu TrpMet Ala Ile Thr Ala Thr Ser His Gly 20 25 30 Ala Arg His Arg Thr Phe ValGly Phe Ser Gly Cys Leu His Arg Lys 35 40 45 Ser Leu Thr Tyr Pro Val IleCys Leu Ser Lys Pro Ser Gln Arg Ile 50 55 60 816 base pairs nucleic aciddouble linear cDNA 22 CTG GAC GAC TCG AAG CGC GTA GCC AAG CGG AAG CTGATC GAG GAG AAC 48 Leu Asp Asp Ser Lys Arg Val Ala Lys Arg Lys Leu IleGlu Glu Asn 1 5 10 15 CGG GAG CGG CGA CGC AAG GAG GAG ATG ATC AAA TCCCTG CAG CAC CGG 96 Arg Glu Arg Arg Arg Lys Glu Glu Met Ile Lys Ser LeuGln His Arg 20 25 30 CCC AGC CCC AGC GCA GAG GAG TGG GAG CTG ATC CAC GTGGTG ACC GAG 144 Pro Ser Pro Ser Ala Glu Glu Trp Glu Leu Ile His Val ValThr Glu 35 40 45 GCG CAC CGC AGC ACC AAC GCG CAG GGC AGC CAC TGG AAG CAGAGG AGG 192 Ala His Arg Ser Thr Asn Ala Gln Gly Ser His Trp Lys Gln ArgArg 50 55 60 AAA TTC CTG CTC GAA GAT ATC GGT CAG TCG CCC ATG GCC TCC ATGCTT 240 Lys Phe Leu Leu Glu Asp Ile Gly Gln Ser Pro Met Ala Ser Met Leu65 70 75 80 GAC GGG GAC AAA GTG GAC CTG GAG GCG TTC AGC GAG TTT ACA AAAATC 288 Asp Gly Asp Lys Val Asp Leu Glu Ala Phe Ser Glu Phe Thr Lys Ile85 90 95 ATC ACG CCG GCC ATC ACC CGC GTG GTC GAC TTT GCC AAA AAC CTG CCC336 Ile Thr Pro Ala Ile Thr Arg Val Val Asp Phe Ala Lys Asn Leu Pro 100105 110 ATG TTC TCG GAG CTG CCG TGC GAG GAT CAG ATC ATC CTG CTG AAG GGC384 Met Phe Ser Glu Leu Pro Cys Glu Asp Gln Ile Ile Leu Leu Lys Gly 115120 125 TGC TGC ATG GAG ATC ATG TCG CTG CGC GCC GCC GTG CGC TAC GAC CCC432 Cys Cys Met Glu Ile Met Ser Leu Arg Ala Ala Val Arg Tyr Asp Pro 130135 140 GAG AGC GAA ACG CTG ACG CTG AGC GGG GAA ATG GCC GTC AAA CGC GAG480 Glu Ser Glu Thr Leu Thr Leu Ser Gly Glu Met Ala Val Lys Arg Glu 145150 155 160 CAG TTG AAG AAC GGA GGG CTG GGG GTC GTG TCT GAT GCC ATC TTCGAC 528 Gln Leu Lys Asn Gly Gly Leu Gly Val Val Ser Asp Ala Ile Phe Asp165 170 175 CTC GGC AAG TCG CTG TCT GCC TTC AAC CTG GAC GAC ACC GAG GTGGCC 576 Leu Gly Lys Ser Leu Ser Ala Phe Asn Leu Asp Asp Thr Glu Val Ala180 185 190 CTG CTG CAG GCC GTG CTG CTC ATG TCC TCA GAC CGG ACG GGG CTGATC 624 Leu Leu Gln Ala Val Leu Leu Met Ser Ser Asp Arg Thr Gly Leu Ile195 200 205 TGC GTG GAT AAG ATA GAG AAG TGC CAG GAG TCG TAC CTG CTG GCGTTC 672 Cys Val Asp Lys Ile Glu Lys Cys Gln Glu Ser Tyr Leu Leu Ala Phe210 215 220 GAG CAC TAC ATC AAC TAC CGC AAA CAC AAC ATT CCC CAC TTC TGGTCC 720 Glu His Tyr Ile Asn Tyr Arg Lys His Asn Ile Pro His Phe Trp Ser225 230 235 240 AAG CTG CTG ATG AAG GTG GCG GAC CTG CGC ATG ATC GGC GCCTAC CAC 768 Lys Leu Leu Met Lys Val Ala Asp Leu Arg Met Ile Gly Ala TyrHis 245 250 255 GCC AGC CGC TTC CTG CAC ATG AAG GTG GAG TGC CCC ACC GAGCTC TCC 816 Ala Ser Arg Phe Leu His Met Lys Val Glu Cys Pro Thr Glu LeuSer 260 265 270 272 amino acids amino acid linear protein 23 Leu Asp AspSer Lys Arg Val Ala Lys Arg Lys Leu Ile Glu Glu Asn 1 5 10 15 Arg GluArg Arg Arg Lys Glu Glu Met Ile Lys Ser Leu Gln His Arg 20 25 30 Pro SerPro Ser Ala Glu Glu Trp Glu Leu Ile His Val Val Thr Glu 35 40 45 Ala HisArg Ser Thr Asn Ala Gln Gly Ser His Trp Lys Gln Arg Arg 50 55 60 Lys PheLeu Leu Glu Asp Ile Gly Gln Ser Pro Met Ala Ser Met Leu 65 70 75 80 AspGly Asp Lys Val Asp Leu Glu Ala Phe Ser Glu Phe Thr Lys Ile 85 90 95 IleThr Pro Ala Ile Thr Arg Val Val Asp Phe Ala Lys Asn Leu Pro 100 105 110Met Phe Ser Glu Leu Pro Cys Glu Asp Gln Ile Ile Leu Leu Lys Gly 115 120125 Cys Cys Met Glu Ile Met Ser Leu Arg Ala Ala Val Arg Tyr Asp Pro 130135 140 Glu Ser Glu Thr Leu Thr Leu Ser Gly Glu Met Ala Val Lys Arg Glu145 150 155 160 Gln Leu Lys Asn Gly Gly Leu Gly Val Val Ser Asp Ala IlePhe Asp 165 170 175 Leu Gly Lys Ser Leu Ser Ala Phe Asn Leu Asp Asp ThrGlu Val Ala 180 185 190 Leu Leu Gln Ala Val Leu Leu Met Ser Ser Asp ArgThr Gly Leu Ile 195 200 205 Cys Val Asp Lys Ile Glu Lys Cys Gln Glu SerTyr Leu Leu Ala Phe 210 215 220 Glu His Tyr Ile Asn Tyr Arg Lys His AsnIle Pro His Phe Trp Ser 225 230 235 240 Lys Leu Leu Met Lys Val Ala AspLeu Arg Met Ile Gly Ala Tyr His 245 250 255 Ala Ser Arg Phe Leu His MetLys Val Glu Cys Pro Thr Glu Leu Ser 260 265 270 25 base pairs nucleicacid double linear DNA 24 TCCCCGGGTA ACTAAGTAAG GATCC 25 24 base pairsnucleic acid double linear DNA 25 AGTGGGTCCC CGGGTGACAT GGAA 24 8 aminoacids amino acid linear polypeptide internal 26 Ser Gly Ser Pro Gly AspMet Glu 1 5 24 base pairs nucleic acid double linear DNA 27 AGTGGGTCCCCGGGTCTGGA CGAC 24 8 amino acids amino acid linear polypeptide internal28 Ser Gly Ser Pro Gly Leu Asp Asp 1 5

1. A non-human transgenic animal having a transgene comprising apolynucleotide sequence encoding a fusion protein which activatestranscription, the fusion protein comprising a first polypeptide whichbinds to a tet operator sequence in the presence of tetracycline or atetracycline analogue operatively linked to a second polypeptide whichactivates transcription in eukaryotic cells.
 2. The animal of claim 1,wherein the first polypeptide of the fusion protein is a mutated Tetrepressor.
 3. The animal of claim 2, wherein the mutated Tet repressorhas at least one amino acid substitution compared to a wild-type Tetrepressor.
 4. The animal of claim 3, wherein the mutated Tet repressoris a mutated Tn10-derived Tet repressor having an amino acidsubstitution at at least one amino acid position selected from the groupconsisting of position 71, position 95, position 101 and position 102.5. The animal of claim 4, wherein the mutated Tn10-derived Tet repressorcomprises an amino acid sequence shown in positions 1 to 207 of SEQ IDNO:
 2. 6. The animal of claim 1, wherein the second polypeptide of thefusion protein comprises a transcription activation domain of herpessimplex virion protein
 16. 7. The animal of claim 1, further having asecond transgene comprising a gene of interest operably linked to atleast one tet operator sequence.
 8. The animal of claim 7, furtherhaving a third transgene comprising a polynucleotide sequence encoding afusion protein which inhibits transcription, the fusion proteincomprising a first polypeptide which binds to a tet operator sequence,operatively linked to a heterologous second polypeptide which inhibitstranscription in eukaryotic cells.
 9. The animal of claim 1, which is amouse.
 10. The animal of claim 1, which is selected from a groupconsisting of a cow, a goat, a sheep and a pig.
 11. A method formodulating transcription of the second transgene in the transgenicanimal of claim 7, comprising administering tetracycline or atetracycline analogue to the animal.
 12. A non-human transgenic animalhaving a transgene comprising a polynucleotide sequence encoding afusion protein which activates transcription, the fusion proteincomprising a first polypeptide which binds to a tet operator sequence inthe presence of tetracycline or a tetracycline analogue operativelylinked to a second polypeptide which activates transcription ineukaryotic cells, wherein the transgene is integrated at a predeterminedlocation within a chromosome within cells of the animal.
 13. The animalof claim 12, wherein the first-polypeptide of the fusion protein is amutated Tet repressor.
 14. The animal of claim 13, wherein the mutatedTet repressor has at least one amino acid substitution compared to awild-type Tet repressor.
 15. The animal of claim 14, wherein the mutatedTet repressor is a mutated Tn10-derived Tet repressor having an aminoacid substitution at at least one amino acid position selected from thegroup consisting of position 71, position 95, position 101 and position102.
 16. The animal of claim 15, wherein the mutated Tn10-derived Tetrepressor comprises an amino acid sequence shown in positions 1 to 207of SEQ ID NO:
 2. 17. The animal of claim 12, wherein the secondpolypeptide of the fusion protein comprises a transcription activationdomain of herpes simplex virion protein
 16. 18. The animal of claim 12,further having a second transgene comprising a gene of interest operablylinked to at least one tet operator sequence.
 19. The animal of claim18, further having a third transgene comprising a polynucleotidesequence encoding a fusion protein which inhibits transcription, thefusion protein comprising a first polypeptide which binds to a tetoperator sequence, operatively linked to a heterologous secondpolypeptide which inhibits transcription in eukaryotic cells.
 20. Amethod for modulating transcription of the second transgene in thetransgenic animal of claim 18, comprising administering tetracycline ora tetracycline analogue to the animal.